Micropropagation and medicinal properties of Barleria greenii

MICROPROPAGATION AND MEDICINAL PROPERTIES OF 

BARLERIA GREENII AND HUERNIA HYSTRIX 

BY 

STEPHEN OLUWASEUN AMOO 

(M.Sc. OBAFEMI AWOLOWO UNIVERSITY, NIGERIA) 

Submitted in fulfilment of the requirements for the degree of 

DOCTOR OF PHILOSOPHY 

Research Centre for Plant Growth and Development 

School of Biological and Conservation Sciences 

University of KwaZulu-Natal, Pietermaritzburg 

November 2009

TABLE OF CONTENTS 

STUDENT DECLARATION ................................................................................... vii 

DECLARATION BY SUPERVISORS ................................................................... viii 

FACULTY OF SCIENCE & AGRICULTURE DECLARATION 1 - PLAGIARISM.... ix 

FACULTY OF SCIENCE & AGRICULTURE DECLARATION 2 - PUBLICATIONS x 

ACKNOWLEDGEMENTS ..................................................................................... xii 

LIST OF FIGURES ............................................................................................... xiii 

LIST OF TABLES .................................................................................................xvii 

LIST OF ABBREVIATIONS .................................................................................. xix 

ABSTRACT….. ....................................................................................................xxii 

Chapter 1 General introduction ........................................................................ 1 

1.1 Use of plants in horticulture and traditional medicine .......................... 1 

1.2 The need for conservation of plant species ........................................... 1 

1.3 Distribution, morphology, uses and conservation status of the 

studied plant species ............................................................................... 3 

1.3.1 Barleria greenii .................................................................................... 3 

1.3.2. Huernia hystrix..................................................................................... 5 

1.4 Value of tissue culture ............................................................................. 7 

1.5 Aims and objectives ................................................................................. 8 

1.6 General overview of the thesis ................................................................ 8 

Chapter 2 Literature review ............................................................................ 10 

2.1 Micropropagation ................................................................................... 10 

2.1.1 Introduction ........................................................................................ 10 

2.1.2 Stage 0: Selection and preparation of mother plants ......................... 10 

2.1.3 Stage I: Initiation and establishment of aseptic culture ...................... 11 

ii

2.1.4 Stage II: Proliferation or multiplication of propagules ......................... 15 

2.1.5 Stage III: Preparation of propagules for transfer to soil ..................... 17 

2.1.6 Stage IV: In vivo rooting and acclimatization for soil establishment... 19 

2.1.7 Effects of auxins and cytokinins on plant regeneration in 

micropropagation ............................................................................. 22 

2.1.8 Environmental factors affecting micropropagation ............................. 29 

2.1.8.1 Light ............................................................................................... 29 

2.1.8.2 Temperature ................................................................................... 31 

2.1.9 Tissue culture of the families: Acanthaceae and Asclepiadaceae ..... 32 

2.2 Pharmacological and phytochemical investigation of plant extracts 36 

2.2.1 Introduction ........................................................................................ 36 

2.2.2 Antimicrobial activity .......................................................................... 37 

2.2.3 Anti-inflammatory activity ................................................................... 39 

2.2.4 Acetylcholinesterase inhibition .......................................................... 42 

2.2.5 Antioxidant activity ............................................................................. 43 

2.2.6 Phytochemical property ..................................................................... 45 

Chapter 3 In vitro propagation of Barleria greenii ........................................ 48 

3.1 Introduction ............................................................................................ 48 

3.2 Materials and methods ........................................................................... 49 

3.2.1 Explant decontamination, selection and bulking ................................ 49 

3.2.2 Effects of BA and NAA on shoot multiplication .................................. 50 

3.2.3 Effects of types and concentrations of cytokinins on shoot 

multiplication .................................................................................... 50 

3.2.4 Effects of photoperiod on shoot multiplication ................................... 51 

3.2.5 In vitro rooting of regenerated shoots ................................................ 51 

3.2.6 Ex vitro rooting and acclimatization ................................................... 52 

3.2.7 Data analyses .................................................................................... 52 

iii

3.3 Results and discussion ......................................................................... 53 

3.3.1 Explant decontamination ................................................................... 54 


3.3.3 Effects of types and concentrations of cytokinins on shoot 

multiplication .................................................................................... 58 

3.3.4 Effects of photoperiod on shoot multiplication ................................... 62 

3.3.5 In vitro rooting of regenerated shoots ................................................ 63 

3.3.6 Ex vitro rooting and acclimatization ................................................... 65 

Chapter 4 In vitro propagation of Huernia hystrix ........................................ 67 

4.1 Introduction ............................................................................................ 67 


4.2.1 Source material, decontamination and bulking of explants ................ 69 


4.2.3 Effects of temperature and photoperiod on shoot multiplication ........ 70 

4.2.4 Determination of titratable acidity ...................................................... 70 

4.2.5 Effects of culture vessel size on shoot multiplication ......................... 71 

4.2.6 Indirect organogenesis ...................................................................... 71 

4.2.7 Rooting and acclimatization ............................................................... 71 

4.2.8 Data analyses .................................................................................... 72 

4.3 Results and discussion ......................................................................... 72 

4.3.1 Explant decontamination ................................................................... 72 

4.3.2 Shoot and root organogenesis .......................................................... 74 

4.3.3 Effects of temperature and photoperiod on shoot multiplication ........ 77 

4.3.4 Titratable acidity ................................................................................ 82 

4.3.5 Effects of culture vessel size on shoot multiplication ......................... 84 

4.3.6 Indirect organogenesis ...................................................................... 86 

4.3.7 Rooting and acclimatization ............................................................... 87 

iv

Chapter 5 Pharmacological and phytochemical evaluation of Barleria 

species and Huernia hystrix ......................................................... 90 

5.1 Introduction ............................................................................................ 90 


5.2.1 Collection of plant materials .............................................................. 91 

5.2.2 Pharmacological evaluation ............................................................... 92 

5.2.2.1 Preparation of extracts ................................................................... 92 

5.2.2.2 Antibacterial activity ....................................................................... 92 

5.2.2.3 Antifungal activity ........................................................................... 93 

5.2.2.4 Anti-inflammatory activity ............................................................... 94 

5.2.2.5 Acetylcholinesterase (AChE) inhibition........................................... 95 

5.2.2.6 Antioxidant activity ......................................................................... 96 

5.2.2.6.1 DPPH radical-scavenging activity ............................................. 96 

5.2.2.6.2 Ferric reducing power activity .................................................... 97 

5.2.2.6.3 β-Carotene linoleic acid assay .................................................. 97 

5.2.3 Phytochemical evaluation .................................................................. 98 

5.2.3.1 Preparation of extracts ................................................................... 98 

5.2.3.2 Total phenolic content .................................................................... 98 

5.2.3.3 Total iridoid content ........................................................................ 99 

5.2.3.4 Flavonoid content ........................................................................... 99 

5.2.3.5 Gallotannin content ...................................................................... 100 

5.2.3.6 Condensed tannin (proanthocyanidin) content ............................. 100 

5.2.4 Data analyses .................................................................................. 101 

5.3 Results and discussion ....................................................................... 101 

5.3.1 Yield of plant extracts ...................................................................... 101 

5.3.2 Pharmacological evaluation ............................................................. 101 

5.3.2.1 Antibacterial activity ..................................................................... 101 

v

5.3.2.2 Antifungal activity ......................................................................... 107 

5.3.2.3 Anti-inflammatory activity ............................................................. 110 

5.3.2.4 Acetylcholinesterase inhibition ..................................................... 112 

5.3.2.5 Antioxidant activity ....................................................................... 115 

5.3.2.5.1 DPPH radical scavenging activity ............................................ 115 

5.3.2.5.2 Ferric ion reducing power activity ............................................ 118 

5.3.2.5.3 β-Carotene linoleic acid assay ................................................ 120 

5.3.3.1 Total phenolic content .................................................................. 124 

5.3.3.2 Total iridoid content ...................................................................... 126 

5.3.3.3 Flavonoid content ......................................................................... 128 

5.3.3.4 Gallotannin content ...................................................................... 130 

5.3.3.5 Condensed tannin (proanthocyanidin) content ............................. 132 

Chapter 6 General conclusions .................................................................... 134 

References ....................................................................................................... 137 

vi

STUDENT DECLARATION 

Micropropagation and medicinal properties of Barleria greenii and 

Huernia hystrix 

I, Stephen Oluwaseun Amoo, Student Number 205527320 

declare that: 

(i) The research reported in this dissertation, except where otherwise 

indicated, is the result of my own endeavours in the Research Centre for 

Plant Growth and Development, School of Biological and Conservation 

Sciences, University of KwaZulu-Natal Pietermaritzburg; 

(ii) This dissertation has not been submitted for any degrees or examination 

at any other University; 

(iii) This thesis does not contain data, figures or writing, unless specifically 

acknowledged, copied from other researchers; and 

(iv) Where I have reproduced a publication of which I am an author or co- 

author, I have indicated which part of the publication was contributed by 

me. 

Signed at Pietermaritzburg on the...…. day of March, 2010. 

__________________________ 

SIGNATURE 

vii

DECLARATION BY SUPERVISORS 

We hereby declare that we acted as Supervisors for this PhD student: 

Student’s Full Name: Stephen Oluwaseun Amoo 

Student Number: 205527320 

Thesis Title: Micropropagation and medicinal properties of Barleria greenii and 

Huernia hystrix 

Regular consultation took place between the student and ourselves throughout the 

investigation. We advised the student to the best of our ability and approved the 

final document for submission to the Faculty of Science and Agriculture Higher 

Degrees Office for examination by the University appointed Examiners. 

__________________________ 

SUPERVISOR: PROFESSOR J VAN STADEN 

________________________ 

CO-SUPERVISOR: DR JF FINNIE 

viii

FACULTY OF SCIENCE & AGRICULTURE DECLARATION 1 - 

I, Stephen Oluwaseun Amoo, declare that 

PLAGIARISM 

1. The research reported in this thesis, except where otherwise indicated, 

is my original research. 

2. This thesis has not been submitted for any degree or examination at any 

other university. 

3. This thesis does not contain other persons‟ data, pictures, graphs or 

other information, unless specifically acknowledged as being sourced 

from other persons. 

4. This thesis does not contain other persons' writing, unless specifically 

acknowledged as being sourced from other researchers. Where other 

written sources have been quoted, then: 

a. Their words have been re-written but the general information 

attributed to them has been referenced 

b. Where their exact words have been used, then their writing has been 

placed in italics and inside quotation marks, and referenced. 

5. This thesis does not contain text, graphics or tables copied and pasted 

Signed 

from the Internet, unless specifically acknowledged, and the source 

being detailed in the thesis and in the References sections. 

……………………………………………………………………………… 

Declaration Plagiarism 22/05/08 FHDR Approved 

ix

FACULTY OF SCIENCE & AGRICULTURE DECLARATION 2 - 

PUBLICATIONS 

PUBLISHED ARTICLES FROM THIS THESIS 

AMOO, S.O., FINNIE, J.F. and VAN STADEN, J. 2009. Effects of 

temperature, photoperiod and culture vessel size on adventitious shoot 

production of in vitro propagated Huernia hystrix. Plant Cell, Tissue and 

Organ Culture 99: 233-238 

Contribution: Experimental work and writing of publication done by the first 

author under the supervision of the last two authors. 

AMOO, S.O., FINNIE, J.F. and VAN STADEN, J. 2009. In vitro 

pharmacological evaluation of three Barleria species. Journal of 

Ethnopharmacology 121: 274-277 




propagation of Huernia hystrix: an endangered medicinal and ornamental 

succulent. Plant Cell, Tissue and Organ Culture 96: 273-278 



CONFERENCE CONTRIBUTIONS FROM THIS THESIS 


pharmacological evaluation of three Barleria species. Joint Conference of 

the 34 th South African Association of Botanists (SAAB) and 7 th Southern 

African Society of Systematic Biology (SASSB). Drakensville Mountain 

Resort, South Africa. (Oral presentation by the first author) 

x

AMOO, S.O., FINNIE, J.F. and VAN STADEN, J. 2009. Micropropagation 

Signed 

of an endangered valuable succulent: Huernia hystrix. 35 th Annual 

Conference of the South African Association of Botanists (SAAB) and 

International workshop on “Phosphate as a limiting resource”. Stellenbosch 

University, South Africa. (Oral presentation by the first author) 

……………………………………………………………………………… 

Declaration Publications FHDR 22/05/08 Approved 

xi

ACKNOWLEDGEMENTS 

I would like to express a special appreciation to my supervisor, Prof J. van Staden 

for his invaluable support, guidance and encouragement throughout the duration 

of this study. I am very grateful for the financial support he provided in the form of 

a postgraduate bursary. 

Many thanks to my co-supervisor, Dr J.F. Finnie, for his constructive advice, as 

well as constant support and encouragement. 

My sincere thanks to all members of the Research Centre for Plant Growth and 

Development, for their extensive support. The administrative staff (Mrs Magnussen 

and Mrs Warren) and my Research Committee members (Dr Bairu and Dr 

Elgorashi) are especially thanked for their support and inputs. My sincere 

appreciation to Dr Gary Stafford and Mrs Alison Young for their help in the 

collection of plants used in this study. Dr Gary Stafford also kindly supplied the 

Huernia hystrix flower used on the cover page. Thanks to my colleagues in the lab, 

especially Ashwell Ndhlala and Mack Moyo, with whom I shared many 

brainstorming sessions related to some aspects of this research. The technical 

assistance provided by Mr Hampton and his team in the use of some equipment 

are much appreciated. 

My special thanks to my parents and brother as well as Grace, Afolad, Folake and 

Bunmi for their understanding, love, support and encouragement. I miss you all. 

Above all, glory, praise and honour to Jehovah God, the omnipotent and 

omniscient one, for his indescribable free gift. 

xii

LIST OF FIGURES 

Chapter 1 

Figure 1.1: Barleria greenii. (A) The plant during flowering (B) Calyx bearing the 

unopened, mature fruits (capsules). ................................................... 4 

Figure 1.2: Flowering Huernia hystrix potted in a small container. Bar = 10 mm. 6 

Chapter 2 

Figure 2.1: Molecular structures of some auxins commonly used in plant tissue 

culture. ............................................................................................. 23 

Figure 2.2: Molecular structures of some cytokinins commonly used in plant 

tissue culture. ................................................................................... 27 

Figure 2.3: The physiological and pathophysiological functions of COX-2 enzyme 

(STEINMEYER, 2000). .................................................................... 41 

Chapter 3 

Figure 3.1: In vitro propagation of Barleria greenii. (A) Stock plant. (B) Control 

(MS medium without PGR). (C) Shoot multiplication on MS medium 

supplemented with 3 µM BA. (D) Shoot multiplication on MS medium 

supplemented with 7 µM MemTR. (E) In vitro rooted regenerated 

shoot ready for acclimatization. (F) Three-month-old fully 

acclimatized plant. Bars = 10 mm. ................................................... 53 

Figure 3.2: Effects of sodium hypochlorite (NaOCl) solution treatments on 

explants decontamination. ............................................................... 54 

Figure 3.3: Effects of BA and NAA on adventitious shoot production of Barleria 

greenii after four weeks of culture. Bars with different letters are 

significantly different (P = 0.05) according to DMRT. ....................... 56 


greenii after six weeks of culture. Bars with different letters are 


Figure 3.5: Effects of half strength MS medium with or without IBA treatment on 

in vitro rooting of regenerated shoots. Bars with different letters are 


xiii

Figure 3.6: Effects of pulsing with different IBA concentrations on ex vitro 

acclimatization of regenerated shoots .............................................. 66 

Chapter 4 

Figure 4.1: In vitro propagation of Huernia hystrix. (A) Stock plant. (B) Control 

with root production (MS medium without plant growth regulators). 

(C) Multiple shoot production accompanied with root formation on MS 

medium supplemented with 5.37 µM NAA and 22.19 µM BA. (D) 

Two-month-old fully acclimatized plants (E) Green callus growth with 

root hairs on MS medium supplemented with 5.37 µM NAA. (F) Root 

regeneration from callus on MS medium supplemented with 2.69 µM 

NAA and 2.22 µM BA. Scale bar = 10 mm. ...................................... 73 

Figure 4.2: Frequencies of explants decontamination in different mercuric 

chloride solution treatments. ............................................................ 74 

Figure 4.3: Nocturnal titratable acidity in Huernia hystrix shoots cultured at 

different temperatures. 22:00, 02:00 and 06:00 h are the beginning, 

middle and end of 8 h dark period respectively. Bars with different 

letters are significantly different (Ρ = 0.05) according to DMRT. ...... 84 

Figure 4.4: Huernia hystrix adventitious shoot production from explants cultured 

in different culture vessels. (A) Screw cap jar [300 ml]. (B) Culture 

tube [40 ml]. Scale bar = 10 mm. ..................................................... 86 

Figure 4.5: Effects of combinations of NAA and BA concentrations on callus 

growth. Bars with different letters are significantly different (Ρ = 0.05) 

according to DMRT. ........................................................................... 87 

Chapter 5 

Figure 5.1: Anti-inflammatory activity of extracts from different parts of three 

Barleria species in COX-1 (on the left side) and COX-2 (on the right 

side) assays. B.p, B.a, and B.g are Barleria prionitis, B. albostellata 

and B. greenii, respectively. (A) and (B) are PE extracts. (C) and (D) 

are DCM extracts. (E) and (F) are EtOH extracts. Bars in the same 

graph bearing different letters are significantly different (P = 0.05) 

according to DMRT. Percentage inhibition by indomethacin in COX-1 

was 63.4 ± 1.98 and COX-2 was 73.6 ± 1.47. ................................ 111 

xiv

Figure 5.2: Anti-inflammatory activity of different extracts of Huernia hystrix. (A) 

COX-1 assay. (B) COX-2 assay. Bars with different letters in each 

graph are significantly different (P = 0.05) according to DMRT. 

Percentage inhibition by indomethacin in COX-1 was 54.73 ± 2.00 

and COX-2 was 63.44 ± 2.52. ........................................................ 112 

Figure 5.3: Dose-dependent acetylcholinesterase inhibition by different parts of 

three Barleria species. B.p = Barleria prionitis, B.g = Barleria greenii, 

B.a = Barleria albostellata. Bars bearing different letters are 

significantly different (P = 0.05) according to DMRT. The AChE 

inhibition activities by galanthamine at 0.5, 1.0 and 2 µM were 49.24, 

59.81 and 77.03%, respectively. .................................................... 113 


Huernia hystrix. Bars bearing different letters are significantly 

different (P = 0.05) according to DMRT. The AChE inhibition 

activities by galanthamine at 0.5, 1.0 and 2 µM were 49.24, 59.81 

and 77.03%, respectively. .............................................................. 114 

Figure 5.5: Dose-dependent curve of DPPH radical scavenging activity of 

different parts of three Barleria species. (A) Leaves (B) Stems (C) 

Roots. ............................................................................................. 116 


different parts of Huernia hystrix. ................................................... 117 

Figure 5.7: Ferric ion reducing power activity of different parts of Barleria 

species. (A) Leaves (B) Stems (C) Roots. ..................................... 119 

Figure 5.8: Ferric ion reducing power activity of different parts of Huernia hystrix. 

....................................................................................................... 120 

Figure 5.9: Antioxidant activities of different parts of three Barleria species in β- 

carotene-linoleic acid model system. Bars bearing different letters in 

each graph are significantly different (P = 0.05) according to DMRT. 

(A) Antioxidant activity (ANT) based on the average β-carotene 

bleaching rate. (B) Oxidation rate ratio (ORR). (C) Antioxidant activity 

(AA) at t = 60 min. (D) Antioxidant activity (AA) at t = 120 min. ...... 122 

Figure 5.10: Antioxidant activities of different parts of Huernia hystrix in β- 

carotene-linoleic acid model system. Bars bearing different letters in 

each graph are significantly different (P = 0.05) according to DMRT. 

xv

(A) Antioxidant activity (ANT) based on the average β-carotene 

bleaching rate. (B) Oxidation rate ratio. (C) Antioxidant activity at 60 

min. (D) Antioxidant activity at 120 min. ......................................... 123 

Figure 5.11: Total phenolic content of different parts of three Barleria species. 

Bars bearing different letters are significantly different (P = 0.05) 

according to DMRT. ....................................................................... 125 

Figure 5.12: Total phenolic content of different parts of Huernia hystrix. Bars 

bearing different letters are significantly different (P = 0.05) according 

to DMRT. ........................................................................................ 125 

Figure 5.13: Total iridoid content of different parts of three Barleria species. Bars 


to DMRT. ........................................................................................ 127 

Figure 5.14: Total iridoid content of different parts of Huernia hystrix. Bars bearing 

different letters are significantly different (P = 0.05) according to 

DMRT. ............................................................................................ 127 

Figure 5.15: Flavonoid content of different parts of three Barleria species. Bars 


to DMRT. ........................................................................................ 129 

Figure 5.16: Flavonoid content of different parts of Huernia hystrix. Bars bearing 


DMRT. ............................................................................................ 129 

Figure 5.17: Gallotannin content of different parts of three Barleria species. Bars 


to DMRT. ........................................................................................ 131 

Figure 5.18: Gallotannin content of different parts of Huernia hystrix. Bars bearing 


DMRT. ............................................................................................ 131 

Figure 5.19: Condensed tannin content (as leucocyanidin equivalents) of different 

parts of three Barleria species. Bars bearing different letters are 

significantly different (P = 0.05) according to DMRT. ..................... 132 

xvi

LIST OF TABLES 

Chapter 2 

Table 2.1: List of some tissue cultured plant species in the Acanthaceae and 

Asclepiadaceae families ..................................................................... 33 

Chapter 3 

Table 3.1: Effects of types and concentrations of cytokinins on adventitious shoot 

production of Barleria greenii ............................................................. 60 

Table 3.2: Effects of photoperiod on adventitious shoot production of Barleria 

greenii after six weeks of culture ........................................................ 63 

Chapter 4 

Table 4.1: Effects of different combinations of NAA and BA on shoot and root 

regeneration of Huernia hystrix after nine weeks of culture ............... 75 

Table 4.2: Frequencies of shoot, root and basal callus production from treatments 

with different concentration combinations of NAA and BA ................. 76 

Table 4.3: Effects of different concentration combinations of NAA and BA on 

fresh weights of adventitious shoots and roots produced per explant 78 

Table 4.4: Effects of temperature and photoperiod on adventitious shoot 

production of Huernia hystrix after eight weeks of culture .................. 79 

Table 4.5: Effects of temperature and photoperiod on frequency and fresh weight 

of regenerated shoots per explant of Huernia hystrix after eight weeks 

of culture ............................................................................................ 80 

Table 4.6: Effects of culture vessel size on adventitious shoot production of 

Huernia hystrix after eight weeks of culture........................................ 85 

Table 4.7: Effects of half-strength MS medium with or without IBA 

supplementation on rooting and acclimatization of regenerated plants 

........................................................................................................... 88 

Chapter 5 

Table 5.1: Yield (% w/w) of extracts prepared from different parts of three Barleria 

species in terms of starting crude material ....................................... 103 

xvii

Table 5.2: Yield (% w/w) of extracts prepared from different parts of Huernia 

hystrix in terms of starting crude material ......................................... 104 

Table 5.3: Antibacterial activity (MIC and MID) of crude extracts from different 

parts of three Barleria species .......................................................... 105 

Table 5.4: Antibacterial activity (MIC and MBC) of crude extracts from different 

parts of Huernia hystrix .................................................................... 106 

Table 5.5: Antifungal activity of crude extracts from different parts of three 

Barleria species against Candida albicans ....................................... 108 

Table 5.6: Antifungal activity of different parts of Huernia hystrix against Candida 

albicans ............................................................................................ 109 

Table 5.7: DPPH radical scavenging activity of different parts of three Barleria 

species ............................................................................................. 117 

Table 5.8: DPPH radical scavenging activity of different parts of Huernia hystrix 

......................................................................................................... 118 

xviii

LIST OF ABBREVIATIONS 

[3G]BA 6-Benzylamino-3-β-D-glucopyranosylpurine 



[9R]BA 6-Benzylamino-9-β-D-ribofuranosylpurine 

2,4-D 2,4-Dichlorophenoxyacetic acid 

AChE Acetylcholinesterase 

AD Alzheimer‟s disease 

AIDS Acquired immune deficiency syndrome 

ANOVA Analysis of variance 

ATCC American type culture collection 

ATCI Acetylthiocholine iodide 

ATP Adenosine triphosphate 

B5 B5 medium (GAMBORG et al., 1968) 

BA 6-Benzyladenine 

BHT Butylated hydroxytoluene (2,6-Di-tert.-butyl-p-cresol) 

CAM Crassulacean acid metabolism 

CNS Central nervous system 

COX Cyclooxygenase 

DCM Dichloromethane 

DHZ Dihydrozeatin 

DIF Difference in photoperiod and dark period temperatures 

DMRT Duncan‟s Multiple Range Test 

DPM Disintegrations per minute 

DPPH 1,1-Diphenyl-2-picrylhydrazyl 

DTNB 5,5-Dithiobis-2-nitrobenzoic acid 

DW Dry weight 

ET Electron transfer 

EtOH Ethanol 

Folin C Folin-Ciocalteu 

FRAP Ferric ion reducing power assay 

FSA Flora of southern Africa 

xix

GAE Gallic acid equivalents 

GST Glutathione S-transferase 

HAT Hydrogen atom transfer 

HE Harpagoside equivalents 

HPLC High-performance liquid chromatography 

IAA Indole-3-acetic acid 

IBA Indole-3-butyric acid 

iNOS Inducible nitric oxide synthase 

INT p-Iodonitrotetrazolium chloride 

iP N 6 -Isopentenyladenine 

IUCN International Union for the Conservation of Nature and 

Natural Resources 

LOX 5-Lipoxygenase 

LTs Leukotrienes 

MBC Minimum bactericidal concentration 

MemTR meta-Methoxytopolin riboside 

MeOH Methanol 

MFC Minimum fungicidal concentration 

MFD Minimum fungicidal dilution 

MH Mueller-Hinton 

MIC Minimum inhibitory concentration 

MID Minimum inhibitory dilution 

MS MURASHIGE and SKOOG (1962) 

mT meta-Topolin 

mTR meta-Topolin riboside 

NAA α-Naphtalene acetic acid 

NADPH Reduced adenine dinucleotide phosphate 

ND Not determined 

NF-кB Nuclear factor-кB 

NSAIDs Nonsteroidal anti-inflammatory drugs 

OG O-glucosides 

OH Hydroxyl 

ORAC Oxygen radical absorbance capacity 

xx

PE Petroleum ether 

PEPC Phosphoenolpyruvate carboxylase 

PGR Plant growth regulators 

PPF Photosynthetic photon flux 

RNA Ribonucleic acid 

ROS Reactive oxygen species 

RSA Radical scavenging activity 

SH Schenk and Hilderbrandt (1972) 

TEAC Trolox equivalent antioxidant capacity 

TLC Thin-layer-chromatographic 

US United States 

WWF World Wildlife Fund 

YM Yeast Malt 

Z trans-Zeatin 

xxi

ABSTRACT 

The crisis of newly emerging diseases and the resistance of many pathogens to 

currently used drugs, coupled with the adverse side-effects of many of these drugs 

have necessitated the continuous search for new drugs that are potent and 

efficacious with minimal or no adverse side-effects. The plant kingdom is known to 

contain many novel biologically active compounds, many of which could potentially 

have a higher medicinal value when compared to some of the current medications. 

Indeed, the use of plants in traditional medicine, especially in African communities, 

is gaining more importance due to their affordability and accessibility as well as 

their effectiveness. Exponential population growth rates in many developing 

countries has resulted in heavy exploitation of our plant resources for their 

medicinal values. In addition, plant habitat destruction arising from human 

developmental activities has contributed to the fragmentation or loss of many plant 

populations. Owing to these factors, many plant species with horticultural and/or 

medicinal potential have become either extinct or are threatened with extinction. 

These threatened species cut across different taxonomic categories including 

shrubs, trees and succulents. Without the application of effective conservation 

strategies, the medicinal and/or horticultural potential of such threatened species 

may be totally lost with time. The extinction of such species could lead to the loss 

of potential therapeutic compounds and/or genes capable of being exploited in the 

biosynthesis of new potent pharmaceutical compounds. 

The overall aims of this study were to establish efficient regeneration protocols 

and explore the medicinal properties of two threatened South African species 

belonging to different taxonomic categories: Barleria greenii (a shrub) and Huernia 

hystrix (a succulent). Barleria greenii is a perennial ornamental and a critically 

endangered shrub, endemic to a small area in KwaZulu-Natal province of South 

Africa. Its conventional propagation is hampered by high seed parasitism and 

difficulty in rooting. Huernia hystrix is a dwarf perennial stem succulent heavily 

exploited for traditional medicine among the Zulu people in South Africa. It is 

considered vulnerable in KwaZulu-Natal province, an endangered endemic to the 

flora of the southern Africa region and is vulnerable in its global conservation 

xxii

status. Being a dwarf species, very limited cuttings can be taken from the mother 

plant for conventional propagation. 

In developing a micropropagation protocol for B. greenii using shoot-tip explants (5 

mm length), the effects of BA with or without NAA combinations, in MS medium 

were evaluated. Concentrations of 0.0, 0.5, and 1.0 µM of NAA were combined 

with 1.0, 2.0, 3.0, 4.0 and 5.0 µM BA in a 3 × 5 completely randomised factorial 

design. The treatments with BA alone gave higher adventitious shoot production 

both after four and six weeks of culture when compared to the BA treatments 

supplemented with NAA concentrations. These results imply that the exogenous 

application of NAA is neither required nor beneficial for adventitious shoot 

induction or proliferation from the shoot-tip explants of this species. In evaluating 

the effects of different types and concentrations of cytokinins on shoot production, 

the treatments with kinetin generally gave a low shoot production whereas BA 

treatments gave increased adventitious shoot production with the optimum at a 

concentration of 3 µM. High abnormality indices were however, observed in all the 

treatments with BA. At higher concentrations (5 and 7 µM), the treatments with 

mTR and MemTR gave increased adventitious shoot production with abnormality 

indices less than that of the control. These results indicated that the topolins (mTR 

and MemTR) are less toxic and more effective in the micropropagation of this plant 

species. The abnormality indices recorded in the topolin treatments could possibly 

be carry-over effects of BA since the explants used were obtained from BA-treated 

cultures. Furthermore, cultures maintained under a 16 h photoperiod gave a 

significantly higher production of adventitious shoots with lengths greater than 10 

mm than those placed under continuous light. Regenerated shoots were ex vitro 

rooted after an IBA pulse-treatment for five hours and acclimatized successfully 

with 65% survival. This developed protocol could potentially produce over 60,000 

transplantable shoots per year from a single shoot-tip explant. 

An efficient and rapid micropropagation protocol was successfully developed for H. 

hystrix. Adventitious shoots were regenerated from stem explants (10 mm length) 

cultured on MS medium supplemented with a range of NAA (0.00, 2.69, 5.37 and 

8.06 µM) and BA (4.44, 13.32 and 22.19 µM BA) concentrations. The treatments 

with a combination of BA and NAA demonstrated a synergistic effect on 

xxiii

adventitious shoot production. A 100% shoot regeneration frequency with a 

production of four adventitious shoots per explant was obtained on MS medium 

containing 5.37 µM NAA and 22.19 µM BA. Callus produced at the base of the 

explant on the same medium showed root organogenic potential. The effects of 

photoperiod and temperature were further evaluated in optimizing this 

micropropagation protocol. Significant increases in shoot proliferation were 

observed with increased temperature in cultures maintained under a 16 h 

photoperiod. Slow growth observed at low temperatures (15 and 20°C) offers a 

potential strategy for cost-effective in vitro storage of H. hystrix germplasm. The 

maximum number of adventitious shoots produced per explant and shoot 

regeneration frequency were observed in cultures maintained at 35ºC, the 

optimum temperature for photosynthesis in plants possessing CAM. The nocturnal 

accumulation of organic acids in cultures incubated under a 16 h photoperiod 

further suggest the presence of CAM in this species. On the other hand, cultures 

kept under continuous light appear to shift to a C-3 photosynthetic pathway. With 

an increase in temperature under continuous light, there was a significant 

decrease in the fresh weight of adventitious shoots regenerated per explant. The 

use of larger culture vessels further increased the shoot proliferation to 5.6 shoots 

per explant with a potential production of 3429 shoots per m 2 in the growth room 

compared to 2750 shoots per m 2 using culture tubes. Regenerated shoots 

produced roots when transferred to half strength MS medium with or without auxin. 

The micropropagated plants were easily acclimatized within two months under 

greenhouse conditions when potted in a soil and sand mixture (1:1; v/v) treated 

with a fungicide (Benlate, 0.01%). More than 95% survival with no observable 

morphological variations was obtained. The developed protocol provides a simple, 

cost-effective means for the conservation of endangered H. hystrix by clonal 

propagation within a short time. 

The dual biological and chemical screening approach was used to evaluate the 

medicinal properties of three Barleria species (including B. greenii) and H. hystrix. 

Different extracts of these species demonstrated antibacterial, antifungal, anti- 

inflammatory, antioxidant and AChE inhibition activities. The observed 

pharmacological activities might be largely due to their relatively high flavonoid 

content, with a contributing effect from their iridoid and tannin compounds. The 

xxiv

activities shown by H. hystrix extracts might possibly explain its heavy exploitation 

in traditional medicine. Extracts from Barleria species generally had comparatively 

higher pharmacological activities and phytochemical content. The concept of 

substituting plant parts (such as leaves and stems for roots) for sustainable 

exploitation was found to be dependent on the species and/or biological activity 

evaluated. The substantial high activities observed with some B. greenii extracts in 

the pharmacological assays used further highlight the need to conserve our plant 

resources before they become extinct, since some of them could be 

pharmacologically active and perhaps contain novel compounds that are 

biologically active against some treatment-resistant infections. 

xxv

Chapter 1 General introduction 

1.1 Use of plants in horticulture and traditional medicine 

Worldwide, there is an ever-growing demand for valuable plants many of which 

are used for horticultural and medicinal purposes. Flowering plant species that can 

be easily potted, are frost-hardy, cold or drought tolerant and requiring little 

maintenance are sought after by collectors due to their high ornamental value. 

Some of these ornamental species are also known to be medicinal, and medicinal 

plants are important in meeting the human need for good healthcare. A report by 

the World Health Organization, for example, shows that about 80% of the world‟s 

population depends on traditional medicines to meet at least some of their primary 

healthcare needs (WHO, 2004). The use of plants in indigenous or traditional 

medicine is more affordable and accessible to most of the population especially in 

African communities, and it is generally believed to be effective. FENNELL (2002) 

observed that about two-thirds of the South African population still use plants for 

traditional medicines. The majority of plants traditionally used for medicinal 

purposes in South Africa are harvested from the wild and are yet to be fully 

analyzed for their bioactive compounds (TAYLOR and VAN STADEN, 2001). In 

addition, the exponential population growth rates in developing countries since the 

latter half of the twentieth century has resulted in increased demand for plant 

resources (JÄGER and VAN STADEN, 2000). This increasing growth rate has 

also resulted in plant habitat destruction to allow for agricultural and settlement 

land among other developmental activities (JÄGER and VAN STADEN, 2000). 

Owing to their over-exploitation, coupled with destructive harvesting methods, 

habitat loss, habitat change and other human activities, many plant species with 

horticultural and/or medicinal potentials have become either extinct or are 

threatened with extinction. 

1.2 The need for conservation of plant species 

According to SARASAN et al. (2006), more than eight thousand plant species 

were added to the International Union for the Conservation of Nature and Natural 

Resources (IUCN) Red List of Threatened Species during the period 1996 -2004. 

1

During this same period, these authors noted that the number of plants recorded 

as „critically endangered‟ increased by over 60%. The International Union for 

Conservation of Nature (IUCN) and the World Wildlife Fund (WWF) estimated that 

up to 60,000 higher plant species could become extinct or nearly extinct by the 

year 2050 if the current trends of utilization continue (ETKIN, 1998). Despite 

increased governmental regulation, destructive and indiscriminate harvesting of 

medicinal plants continues unabated especially in Africa, where the collection of 

medicinal plants mainly from the wild has become a form of rural self-employment 

(AFOLAYAN and ADEBOLA, 2004). The rising rate of unemployment coupled 

with the recent global economic recession might even worsen the current situation. 

Although South Africa is very rich in floral biodiversity, many of the species are 

highly endemic, heavily exploited and are thus facing the risk of becoming extinct. 

Added to this problem is the fact that some of the species involved are slow- 

growing, not readily cultivated and with a high habitat specificity. These threatened 

species cut across different taxonomic categories including shrubs, trees and 

succulents. 

In the light of the current increasing demands (which far exceeds supply), the 

medicinal and/or horticultural potentials of such species may be totally lost if 

efforts are not geared towards their conservation. Their extinction would mean, 

amongst other things, the loss of genes which could be used for plant 

improvement or in the biosynthesis of new compounds (RATES, 2001). It would 

also mean the loss of interesting chemical compounds (RATES, 2001) with 

pharmaceutical or nutraceutical potential. CUNNINGHAM (1993) observed that 

the majority of plants used in traditional medicines have not been adequately 

screened for active ingredients. He therefore suggested that conservation efforts 

be directed at all species vulnerable to being over-exploited. Moreover, the 

conservation of plant species is crucial to the survival of other life-forms since 

plants contribute to the integrity of our environment (ABOEL-NIL, 1997). For 

instance, it has been estimated that a disappearing plant (due to extinction) can 

take with it ten to thirty other species such as insects, higher animals, and even 

plants that depend directly or indirectly on it (WOCHOK, 1981). 

2

1.3 Distribution, morphology, uses and conservation status of the studied 

plant species 

1.3.1 Barleria greenii 

The genus Barleria, belonging to the Acanthaceae family, is a large genus of 

herbs and shrubs comprising about 300 species worldwide (MAKHOLELA et al., 

2003). The richest representation is in Africa where there are two centers of 

diversity, one in tropical east Africa (about 80 species) and the other in southern 

Africa (about 70 species) (BALKWILL and BALKWILL, 1998). Most species in 

this genus show a high degree of regional endemism. For example, the Indian 

subcontinent, West Africa, southern Africa and East Africa are reported to have 

75, 72, 65 and 56% endemism, respectively (BALKWILL and BALKWILL, 1998). 

Barleria greenii M.-J. Balkwill & K. Balkwill is one such species endemic to South 

Africa. The first population of B. greenii was discovered in 1984 by Dave Green, a 

farmer and amateur botanist from the Estcourt district of Natal (BALKWILL et al., 

1990). It is extremely localized and extremely restricted in distribution, occurring in 

eight localities on three farms near Estcourt, South Africa (MAKHOLELA et al., 

2003). Plants belonging to this species are found in open, rocky areas on 

moderately sloping north-facing aspects, mostly between the 1200 m and 1260 m 

contours (BALKWILL et al., 1990). The ballistic seed dispersal occurring over 

short distances further affects its distribution such that long-range dispersal to new 

suitable habitats occurs rather rarely (BALKWILL et al., 1990). 

Barleria greenii is a perennial, profusely branched woody shrub (Figure 1.1) up to 

1.8 m high (BALKWILL et al., 1990). Its growth form is affected by light intensity 

and the frequency with which its habitat area is burnt (BALKWILL et al., 1990). 

BALKWILL et al. (1990), for example, observed that plants growing in the shade 

are much less robust and have broader leaves than those growing in full sun. They 

noted that plants burnt less often are extremely robust, woody, attaining heights of 

almost 2 m, whereas those burnt often are less robust, attaining a greater 

diameter with more vigorous branching but a height of only 0.8 m. Barleria greenii 

flowers from mid-to late summer, usually over a period of a few weeks 

(MAKHOLELA et al., 2003). The attractive flowers, ranging from pure white to 

3

dark pink with magenta streaks on the corolla lobes, emit a strong, sweet 

fragrance at night and produce large quantities of nectar (MAKHOLELA et al., 

2003). The fruits, which are produced from early to late autumn, are a 4-seeded 

capsule, green when young, black when mature (Figure 1.1) and dehiscing 

explosively (BALKWILL et al., 1990). The seeds are discoid, greyish-black, and 

covered in hygroscopic hairs (BALKWILL et al., 1990). 

Barleria greenii is a beautiful garden plant, flowering prolifically. It grows under a 

wide range of conditions and is frost hardy (TURNER, 2001). SCOTT-SHAW 

(1999) described it as a successful and popular garden plant with attractive 

flowers. Other Barleria species grown as ornamentals include B. cristata, B. 

repens and B. prionitis. Although B. greenii has no recorded usage in traditional 

medicine, many Barleria species have been reportedly used in folk medicine and 

validated to contain compounds possessing biological effects such as anti- 

inflammatory, analgesic, antileukemic, antitumor, antihyperglycemic, anti-amoebic, 

virucidal and antibiotic activities. 

Figure 1.1: Barleria greenii. (A) The plant during flowering (B) Calyx bearing 

the unopened, mature fruits (capsules). 

4

Out of eighteen Barleria species listed by HILTON-TAYLOR (1996) in the Red 

Data List of Southern Africa plants, thirteen are endemic to the flora of southern 

Africa (FSA) region. These include Barleria natalensis (extinct in its global 

conservation status), B. argillicola and B. greenii (global conservation status: 

vulnerable), as well as B. dolomiticola and B. solitaria (global conservation status: 

rare). SCOTT-SHAW (1999) described B. greenii as a very rare species with 

narrow distribution, low abundance and high habitat specificity, and endangered in 

its conservation status. Currently, B. greenii is listed as „critically endangered‟ in 

the National Red List of South African plants (SANBI, 2009). 

1.3.2. Huernia hystrix 

Huernia (Family: Asclepiadaceae) is a genus of about sixty-four species found in 

eastern and southern Africa, Ethiopia and Arabia (HODGKISS, 2004). HILTON- 

TAYLOR (1996) listed sixteen Huernia species including H. hystrix as endemic to 

the FSA region. Huernia hystrix has three varieties, which are hystrix, nova, and 

parvula. This succulent species is reported to be very rare, of narrow distribution 

and low abundance, especially the parvula variety (SCOTT-SHAW, 1999). 

The plants are dwarf, perennial stem succulents which are normally mat forming or 

creeping, rarely pendulous (LIEDE-SCHUMANN and MEVE, 2006). The grey- 

green, five-ridged, prickled stems often branch from the base (Figure 1.2) forming 

large clusters. Their attractive flowers with short stalks have a five-angled margin 

or are five-lobed with a characteristic small lobe in the angle between the main 

lobes (HODGKISS, 2004). The plants are free-flowering in late summer; the 

flowers being quite frilly and particularly attractive (HODGKISS, 2004). 

Huernia hystrix is in the category of Cactus and succulent plants. Being drought- 

tolerant, it is suitable for xeriscaping and can easily be grown in small containers. 

AL-TURKI (2002) described it as an excellent ornamental for rock gardens. 

Huernia species (likely H. hystrix) are reportedly consumed as typical famine-food 

plants in southern Ethiopia (GUINAND and LEMESSA, 2000). According to 

SCOTT-SHAW (1999), the whole plant of H. hystrix is heavily exploited for 

5

traditional medicine (muthi) among the Zulu people in South Africa, though an 

assessment of its medicinal usage has to be done. It is traded in traditional 

medicine system and commonly known as toad plant or ililo elinsundu (Zulu). The 

infusions of the plant are reportedly used as protective charms (HUTCHINGS et 

al., 1996). In Swaziland, the stem of H. hystrix is said to be used for sexual 

stimulation (LONG, 2005). Other Huernia species reported to be medicinal include 

H. stapelioides and H. zebrina, though their medicinal usages were not specified 

(LONG, 2005). 

Of the sixteen endemic Huernia species in the Red Data List of southern African 

plants, two are extinct, one endangered, two vulnerable, and five rare (HILTON- 

TAYLOR, 1996). Huernia hystrix is considered vulnerable in KwaZulu-Natal, 

endangered endemic to FSA region and vulnerable in its global conservation 

status (HILTON-TAYLOR, 1996; SCOTT-SHAW, 1999). It is threatened by its 

heavy exploitation for traditional medicine. Its collection or harvesting is destructive 

since the whole plant is often used. Furthermore, the plants are susceptible to 

stem and root mealy bugs, and damage from these may well facilitate fungal 

attack (HODGKISS, 2004). 

Figure 1.2: Flowering Huernia hystrix potted in a small container. Bar = 10 mm 

6

1.4 Value of tissue culture 

The application of tissue culture as a biotechnological tool in the conservation of 

threatened economic plants has gained tremendous impetus in the last two 

decades. The tissue culture technique is a powerful tool for plant germplasm 

conservation and can be a viable alternative to conventional propagation of slow 

growing species or species that produce recalcitrant or few viable seeds (ABOEL- 

NIL, 1997). Rapid and mass propagation of plant species and their long-term 

germplasm storage are achievable in a small space and short time, with no 

damage to the existing population. Plant material can be produced throughout the 

year without any seasonal limitation. Due to the aseptic nature of tissue culture 

technique, large numbers of uniform and disease-free plants can be produced 

from very small portions of the mother plant. The sterile nature of in vitro cultures 

facilitates the exchange of germplasm or plant materials even at international level 

(SALIH et al., 2001). 

Furthermore, plant tissue culture systems often serve as model systems in the 

study of physiological, biochemical, genetic and structural problems related to 

plants (TORRES, 1989). The use of in vitro culture technique has found 

applications in genetic breeding and improvement, production of new hybrids and 

overcoming incompatibility during crosses. Somaclonal variation, often referred to 

as „off-types‟ is sometimes considered to be an unwanted side-effect of in vitro 

culture technique. However, depending on the research objective, this could be 

advantageous in increasing the pool of variability. The variability can result in 

superior plants or plants with adaptive advantage that can be selected and further 

exploited in genetic improvement programs. Moreover, the control of chemical and 

physical conditions of in vitro cultures makes it possible to optimize conditions 

needed for enhanced production of secondary metabolites in target cells or tissues 

(ABOEL-NIL, 1997). In vitro production of secondary metabolites, in turn, offers a 

steady and reliable source of supply for pharmaceutical or nutraceutical industries 

(GIULIETTI and ERTOLA, 1999). 

7

1.5 Aims and objectives 

Endemic and threatened South African species fall in different taxonomic 

categories including succulents, shrubs and trees. The overall aims of this study 

were to establish efficient regeneration protocols and explore the medicinal 

properties of two threatened South African species belonging to different 

taxonomic categories: Barleria greenii (a shrub) and Huernia hystrix (a succulent). 

The specific objectives were to: 

Determine the appropriate chemical (nutritional) and environmental 

conditions for in vitro propagation of each of the two species; 

Investigate the acetylcholinesterase inhibition, antimicrobial, anti- 

inflammatory and anti-oxidant activities of different extracts of these 

species; 

Investigate the possibility of plant-part substitution as a conservation 

strategy against destructive harvesting of these species for medicinal 

purpose; 

Explore the phytochemical properties of different parts of these species. 

1.6 General overview of the thesis 

Chapter 2 provides background information from literature, relating to 

micropropagation techniques and major factors affecting the success of the 

technique. It also provides an insight into the fundamental principles underlying the 

pharmacological activities evaluated in this study. 

Chapter 3 gives an insight into the development of the micropropagation protocol 

established for B. greenii. The results suggest that exogenous application of NAA 

is neither required nor beneficial for in vitro shoot induction or multiplication from 

shoot-tip explants of this species. The effects of types and concentrations of 

aromatic cytokinins as well as the effects of different light periods were 

investigated and will be discussed. 

8

Chapter 4 describes the simple, rapid and cost-effective clonal propagation 

protocol developed for H. hystrix. The findings indicate that both BA (cytokinin) 

and NAA (auxin) have a synergistic effect on shoot multiplication from stem 

explants of this species. The chapter further highlights the need to investigate the 

effects of environmental conditions when developing efficient micropropagation 

protocols, especially for commercial purposes. Optimizing environmental 

conditions could increase growth rate, reduce labour costs and thus subsequent 

production costs. 

Chapter 5 presents the pharmacological and phytochemical properties of extracts 

from different parts of B. greenii and H. hystrix. For comparison purpose, extracts 

from two other Barleria species (B. albostellata and B. prionitis) were included. 

The results indicate the medicinal potential of the studied plants. Different extracts 

of different plant parts show different potencies in the activities evaluated. The 

possibility of plant part substitution as a conservation measure is discussed. 

Chapter 6 summarizes the major conclusions drawn from this study. 

The section „References‟ provides an alphabetical list of publications or materials 

cited in this thesis. 

9

2.1 Micropropagation 

2.1.1 Introduction 

Chapter 2 Literature review 

The term „micropropagation‟ also known as „in vitro propagation‟ generally involves 

aseptic culturing of excised small plant parts (explants) in an artificial medium 

under appropriate physical conditions for the purpose of producing clonal plants 

capable of surviving in a natural environment. Although it is not different in 

principle from the conventional propagation of some species by cuttings, the use 

of smaller propagules, provision of aseptic and controlled environmental 

conditions, heterotrophic development, and faster plant multiplication makes the 

micropropagation technique more effective (MURASHIGE, 1978). The process of 

micropropagation is often accomplished in different but sequential stages. 

MURASHIGE (1978) proposed four widely accepted stages (I – IV) while 

DEBERGH and MAENE (1981) suggested an additional stage 0. These stages 

are often used worldwide in research and commercial laboratories for plant 

propagation through tissue culture. The objectives of each of the stages and what 

they involve are discussed in the following sections. 

2.1.2 Stage 0: Selection and preparation of mother plants 

The objective of this first stage in micropropagation is to select mother (stock) 

plants that could provide healthy explants capable of being uniformly initiated into 

culture (DEBERGH and MAENE, 1981). Plants that are healthy, apparently free 

from any disease symptom and vigorously growing are often selected as stock 

plants (CONSTABEL and SHYLUK, 1994). In some cases, however, selected 

stock plants are preconditioned by growing them in the greenhouse at a relatively 

low humidity and avoiding overhead watering (DEBERGH and MAENE, 1981). In 

addition, subjecting the stock plants sometimes to antibiotic, fungicidal and 

antiviral treatments could help reduce the contamination level of explants taken 

subsequently from them (MURASHIGE, 1978). In any case, the careful selection 

10

and preparation of stock plants would not only allow for the availability of 

standardized and healthy explants, but also increase the rate of explant survival 

during culture initiation (DEBERGH and MAENE, 1981). 

2.1.3 Stage I: Initiation and establishment of aseptic culture 

Owing to the inherent totipotentiality of plant cells (MURASHIGE, 1978), explants 

obtained from different plant tissues have a potential to regenerate plantlets when 

given appropriate environmental and nutrient conditions. It is important that the 

explants be able to grow well in an aseptic in vitro environment free from any 

obvious infection (MURASHIGE, 1974). In order to achieve this objective, the 

choice of explants and their decontamination are given serious attention during 

this stage. 

MURASHIGE (1974) listed five important factors to be considered when choosing 

suitable explants. These include (i) the organ serving as explant source; (ii) the 

physiological or ontogenic age of the organ; (iii) the season in which the explant is 

obtained; (iv) the size of the explant; and (v) the overall quality of the stock plant. 

Nearly all plant organs or tissues can serve as a source of explants in 

micropropagation. However, explants taken from different plant parts often 

manifest different regeneration and morphogenic responses. KOMALAVALLI and 

RAO (2000), for example, reported different propagation rates and morphogenic 

responses in axillary node, shoot-tip, cotyledonary node, leaf, petiole, root and 

internode explants of Gymnema sylvestre. They observed that only axillary node, 

cotyledonary node and shoot-tip explants readily regenerated multiple shoots 

while other explants produced only callus. These observations were attributed to 

the variation in endogenous levels of plant growth regulators in the explants at the 

time of excision. Similar results have been reported by other researchers in 

different plant species (NHUT et al., 2004; ISLAM et al., 2005; CONDE et al., 

2008). 

The regenerative and morphogenic capacities of explants are sometimes affected 

by the physiological age of the explant and the degree of differentiation among 

11

their constituent cells (MURASHIGE, 1974). SUDHA et al. (1998), while 

investigating the regeneration potential of shoot-tip, terminal and basal node 

explants in Holostemma annulare, observed more rapid multiple shoot formation 

from basal node explants. They concluded that this variation in regenerative 

response might be due to the differences in endogenous growth regulator level, 

nutrient availability and physiological status of the explants. In general, explants 

obtained from juvenile plants often have a better regenerative capacity compared 

to those from older plants, especially in trees and shrubs (NHUT et al., 2007), 

perhaps due to their meristematic cell types and other endogenous factors 

(FENNELL, 2002). BECERRA et al. (2004) reported an inverse linear 

regeneration capacity in relation to the age of donor plants in Passiflora edulis. 

They noted that leaf explants taken from juvenile plants (1- to 6-month-old) 

showed a statistically significant higher shoot regeneration compared to explants 

taken from reinvigorated adult plants (shoots emerging next to the base after 

severe pruning of 1-year-old adult plants). Even among the juvenile explant 

sources, the same inverse linear regeneration capacity with age was observed. In 

Gerbera jamesonii, flower bud age was reported to significantly affect the survival 

rate and shoot regeneration capacity of the receptacle transverse thin cell layers, 

with the optimum observed in 10-day-old flower buds (NHUT et al., 2007). The 

authors further observed that the position of receptacle transverse thin cell layers 

significantly affected the receptacle morphogenic ability. They noted that shoot 

production and regeneration frequency decreased from the middle to exterior 

receptacle layers, possibly due to more nutrient reserves in the middle layers. 

The regenerative capacity of explants in tissue culture could also be affected by 

the season in which the explant is obtained due to seasonal influences on the 

plant developmental stages. PRAKASH and VAN STADEN (2008) observed that 

offshoots of Searsia dentata collected during the vegetative stage (April - May) 

exhibited a better morphogenic response compared to those collected during the 

flowering stage (October – November). Similarly, young offshoots of Hoslundia 

opposita collected during the months of March and April were reported to elicit a 

better morphogenic response compared to those collected during other months of 

the year (PRAKASH and VAN STADEN, 2007). Other researchers have reported 

12

similar differential seasonal responses in explants of different plant species (LITZ 

and CANOVER, 1981; RAZDAN, 2003). 

The size of explants has been reported to influence their survival and growth rates 

in vitro. Very small shoot-tip explants such as meristem tips are said to have a low 

survival and initial growth rates (RAZDAN, 2003) and are therefore not practical 

for achieving rapid clonal multiplication (MURASHIGE, 1974). Nevertheless, these 

authors noted that very small explants (such as submillimetre shoot-tips) are very 

useful in obtaining virus-free plants from an infected individual. STROSSE et al. 

(2008) observed that 5 mm sized explants of banana produced two to three times 

more shoots in 3-month-old cultures than 1 mm sized shoot-tips. On the other 

hand, OTHMANI et al. (2009) reported a decrease in embryogenic callus 

frequency in date palm with an increase in explant size. They observed that leaf 

explants (5 – 10 mm) had the highest embryogenic callus frequency while in larger 

explants (15 – 20 mm), the peripheral parts either turned brown or produced 

hyperhydric non-embryogenic callus. A similar observation was reported in 

Triticum turgidum with small explants showing a higher morphogenic capacity 

(BENKIRANE et al., 2000). According to the authors, larger explants have more 

normal tissue interactions than the smaller ones and such interactions have a 

tendency to inhibit cell division. 

As regards the overall quality of the donor plants, explants obtained from healthy 

plants are generally known to respond better than those from diseased plants 

(MURASHIGE, 1974). LIU and PIJUT (2008) observed that only selected leaf 

explants from Prunus serotina shoots in an active state of growth and with no sign 

of chlorosis regenerated adventitious shoots with a high frequency. MURASHIGE 

(1974) noted that an explant taken soon after heavy fertilization of its source plant 

may produce a different response in the same culture medium compared with 

explants taken later from the same plant or from an unfertilized plant. Such 

differential responses could be largely due to the differences in the physiological 

status of the parent plants. 

In addition to making a good choice of explants, the decontamination treatments 

given to the explants are crucial for their successful initiation into culture. Since the 

13

media are sterile, the explants mainly need to be free of contaminants in order to 

establish an aseptic culture. Culture contamination often results in explant death 

as contaminants rapidly outgrow the cultured explants, exploit the nutrient-rich 

medium, starve the explants, or even produce phytotoxic substances. Surface- 

decontamination of explants is often done by soaking the explants in „sterilants‟ 

such as sodium hypochlorite, calcium hypochlorite, hydrogen peroxide, silver 

nitrate and mercuric chloride solutions for some seconds or minutes, followed by 

thorough rinsing with sterile distilled water under aseptic conditions (TORRES, 

1989). MURASHIGE (1978) highlighted other measures that could enhance the 

effectiveness of the disinfectant. These include the addition of small quantities of 

detergent (such as Tween 20 which acts as a surfactant), performing the 

disinfestations under gentle vacuum or with constant and relatively vigorous 

agitation and prewashing the plant material with alcohol or detergent. In a situation 

where the explant is internally infested by bacteria, fungi or viruses, the inclusion 

of antibiotics, fungicides or anti-viral agents respectively in the preliminary culture 

media has been suggested (MURASHIGE, 1978; PEIXOTO et al., 2006). 

Another problem usually experienced during culture initiation of some explants is 

explant browning, which often results in necrosis or tissue death. Browning of 

explants is most severe in high-tannin or other hydroxyphenol containing species 

(TORRES, 1989). The browning is attributed to the action of copper-containing 

oxidase enzymes (such as polyphenol oxidase and tyrosinase), which are 

produced and/or released due to wounding during the excision of the tissue 

(TORRES, 1989). The incorporation of antioxidants (such as ascorbic or citric 

acid), and/or absorbents (such as activated charcoal or polyvinylpyrrolidone) into 

the culture medium as well as regular subculturing to a fresh medium, culturing in 

the dark, or excising the explant tissues under sterile distilled water are various 

suggested measures to minimize or prevent this problem (RAZDAN, 2003). 

Although the use of activated charcoal is said to be inhibitory in some cultures due 

to its absorption of plant growth regulators, its stimulatory effect has nevertheless 

been reported in some other cultures (TORRES, 1989; RAZDAN, 2003). 

14

2.1.4 Stage II: Proliferation or multiplication of propagules 

This stage aims at optimizing the production of propagules which can potentially 

give rise to whole plants. Multiplication can be brought about by axillary shoot 

production, adventitious shoot proliferation as well as asexual or somatic 

embryogenesis. The latter two regeneration methods can occur either directly from 

the cultured explants or indirectly via callus production. Although a particular 

predetermined regeneration pathway may be inherent within a specific tissue, the 

type and concentration of exogenous plant growth regulators as well as the culture 

environmental conditions often affect and can modify the expression of a 

regeneration method. In deciding the most appropriate multiplication method, 

however, factors such as the rate and frequency of multiplication as well as the 

probability of producing aberrant plants should be seriously considered 

(MURASHIGE, 1978). 

During axillary shoot proliferation, axillary and terminal buds (quiescent or active) 

from the nodal or shoot-tip segments cultured on appropriate media grow into 

axillary shoots. The newly produced shoots with buds along their axis can, in turn 

be used to regenerate more shoots until a satisfactory number is achieved. 

Although the axillary shoot proliferation method is considerably slower than other 

multiplication methods, it allows a yearly multiplication rate that is much faster than 

conventional propagation by cuttings (MURASHIGE, 1978). In addition, it has 

great potential for shoot multiplication in woody plant species that could not be 

successfully regenerated through adventitious shoot proliferation and somatic 

embryogenesis (TORRES, 1989). Regeneration through axillary shoot production 

has been reported in many plant species such as Opuntia spp. (GARCÍA- 

SAUCEDO et al., 2005), Cedrela fissilis (NUNES et al., 2002) and Chimonanthus 

praecox (KOZOMARA et al., 2008), which are succulents, a tree and a shrub, 

respectively. 

Adventitious shoots can be regenerated from nearly all plant tissues or organs 

either directly or indirectly (via callus production). They often develop from sites 

where meristems do not exist (RAZDAN, 2003). Whereas this regeneration 

method is more rapid, the rate of producing genetically or epigenetically altered 

15

plants is higher (MURASHIGE, 1978) especially when the regeneration is indirect 

through callus production. Minimizing the number of subcultures, especially on 

media containing plant growth regulators, is therefore encouraged since both the 

loss of morphogenic capacity and abnormality or variation frequency increase 

progressively with each subculture (MURASHIGE, 1978; TORRES, 1989; BAIRU 

et al., 2008). Nevertheless, the maintenance of regenerative ability, for example, 

of a 5-year-old callus subcultured at 6-weekly intervals has been reported (SATO 

et al., 1995). Some researchers have recorded simultaneous shoot multiplication 

through axillary and adventitious pathways in some cultures (PIERIK, 1987; DE 

FÁTIMA et al., 1996; NUNES et al., 2002; DÁVILA-FIGUEROA et al., 2005; 

PRAKASH and VAN STADEN, 2008). 

Somatic embryogenesis involves the production of embryos from vegetative or 

somatic cells. These embryos could, in turn develop into whole plants. Direct 

somatic embryogenesis occurs when an embryo develops directly from a cell or 

tissue without the callus phase. The cells giving rise to the embryos, in this case, 

are said to be pre-embryonic determined (PIERIK, 1987). On the other hand, an 

unorganized mass of relatively undifferentiated cells (callus) with proembryogenic 

masses may be formed from an explant (SRIVASTAVA, 2002). When subcultured 

on media without auxin, these proembryogenic masses could develop through 

different embryo stages into whole plants. Somatic embryogenesis is said to have 

the greatest potential for achieving rapid clonal micropropagation (TORRES, 

1989). It has major application in genetic transformation and biolistic gene transfer 

(OTHMANI, et al., 2009). In addition, the formation of synthetic seeds through 

somatic embryogenesis could facilitate the development of compact storage, 

packaging and distribution methods for superior varieties (PAREEK and 

KOTHARI, 2003). However, somatic embryogenesis is not often used in practice 

as a means of propagation due to some problems as listed by PIERIK (1987) and 

OTHMANI et al. (2009). These problems include the recalcitrance of some 

species to this method, the high probability of developing mutations, abnormal 

development, low maturation and germination frequencies of somatic embryos, 

and the increasing probability of losing regenerative capacity with repeated 

subculture. Nevertheless, many researchers have reported successful 

regeneration through direct or indirect somatic embryogenesis from explants of 

16

different plant species (GILL et al., 1995; GOGATE and NADGAUDA, 2003; 

PAREEK and KOTHARI, 2003; GOMES et al., 2006; RAI et al., 2007; AZAD et 

al., 2009; OTHMANI et al., 2009). 

2.1.5 Stage III: Preparation of propagules for transfer to soil 

Regenerated shoots or plantlets produced during the immediate previous stage 

(stage II) are often too small or of low vigour to be able to survive in soil. The 

objective of this third micropropagation stage is therefore to prepare regenerated 

shoots or plantlets for successful transfer to soil (MURASHIGE, 1978). This stage 

involves the in vitro rooting of individual regenerated shoots, hardening of plants to 

impart some disease resistance and tolerance to moisture stress, as well as 

rendering the plants capable of autotrophic development (MURASHIGE, 1978). 

Although in vitro rooting is generally an expensive, labour-consuming process, 

especially from a commercial perspective, it may be the only practical method of 

rooting plantlets in some species (TORRES, 1989). DE KLERK (2002) reported 

that in vitro rooting of apple microcuttings strongly increased plantlet size during 

the rooting treatment, enhanced acclimatization by improved water uptake during 

early acclimatization and facilitated the addition of extra nutrients and protective 

compounds that may strongly improve performance. In vitro rooting is often 

achieved by culturing shoot cuttings on medium with half-strength concentrations 

of macro- and micronutrients, with or without auxins (TORRES, 1989; 

MONCOUSIN, 1991). This however, is often dependent on the plant species. SUN 

et al., (2008), for example, observed a significantly higher rooting frequency in 

Zygophyllum xanthoxylon shoots cultured on full-strength MS medium compared 

to those cultured on half-strength medium. On the other hand, PATIL (1998) 

reported an inverse relationship between media strength and rooting frequency in 

three different species of Ceropegia. Indeed, many other researchers have 

reported better in vitro rooting on low-strength media compared to full-strength 

(SAXENA and BHOJWANI, 1993; PUROHIT et al., 1994; ANDRADE et al., 

1999; BEENA et al., 2003). The beneficial effect of low medium strength on 

rooting has been reported to be possibly due to a reduced requirement of nitrogen 

by the plantlets for root formation (HYNDMAN et al., 1982). In some plant species, 

17

adventitious roots are produced without the application of exogenous auxin. This 

response is reportedly triggered by the endogenous auxin produced in the shoot 

apex and transported basipetally to the cut surface (DE KLERK et al., 1999). 

NORDSTRÖM and ELIASSON (1991) observed that the removal of the apex in 

pea cuttings resulted in a reduction of both the endogenous auxin level in the 

basal part of the cutting as well as the number of roots produced. 

In situations where auxins are exogenously applied for rooting purposes, the 

auxins frequently added to the media include IAA (0.1 – 10.0 mg l -1 ), IBA (0.5 – 3.0 

mg l -1 ) and NAA (0.05 – 1.0 mg l -1 ) (TORRES, 1989). BEENA et al. (2003) 

observed that IBA was the best for inducing roots in Ceropegia candelabrum, 

followed by IAA whereas treatments with NAA showed callus formation and poor 

root induction. Similarly, CHOFFE et al. (2000) reported that IAA was less 

effective than IBA for induction of root organogenesis from Echinacea purpurea 

hypocotyl explants, while treatments with NAA were ineffective. The superiority of 

IAA and IBA over NAA was also reported in Metrosideros excelsa (IAPICHINO 

and AIRÒ, 2008). According to FOGAÇA and FETT-NETO (2005), the efficacy of 

IBA in inducing adventitious rooting in Eucalyptus globulus and E. saligna might be 

due to its capacity to be converted to IAA (EPSTEIN and LAVEE, 1984), thus 

serving as a slow release reservoir of a more easily metabolized auxin. 

Conversely, these authors attributed the negative response of NAA to its longer 

persistence or stability in plant tissues, resulting in inhibition of root growth. DE 

KLERK et al. (1997) suggested that NAA might be the preferable auxin 

(particularly when applied as a short term initial treatment) for rooting plants with a 

high activity of auxin-oxidase, due to the non-destruction of NAA by auxin-oxidase. 

High auxin concentrations are generally known to stimulate root induction but 

inhibit root initiation or emergence (DE KLERK et al., 1997, 1999). Ultimately, the 

effectiveness of auxins in rooting processes depends on the affinity of the auxin 

receptor involved in rooting and the concentration of free auxin that is reached in 

the target cells (DE KLERK et al., 1999). The authors stated further that the 

concentration reached in the target cells depends on the amount of endogenous 

auxin as well as the uptake, transport and metabolism of the exogenously applied 

auxin. 

18

Other medium additives such as phloroglucinol and activated charcoal could be 

beneficial in the rooting of some species. BOPANA and SAXENA (2008), for 

instance, observed that the inclusion of phloroglucinol was critical in the rooting of 

Asparagus racemosus, as it enhanced the rooting frequency considerably. 

According to these authors, phloroglucinol acts as an auxin synergist during the 

auxin sensitive phase of root induction. The root-promoting effects of activated 

charcoal have been extensively reviewed by PAN and VAN STADEN (1998). 

In addition to reducing nutrient level, hardening and preparation of plants for 

autotrophic development can be accomplished by the use of growth retardants, by 

reducing relative humidity as well as increasing the photosynthetic photon flux and 

the carbon dioxide concentration in cultures (MURASHIGE 1978; DEBERGH, 

1991; HAZARIKA, 2003). Measures for reducing relative humidity as listed by 

HAZARIKA (2003) in his review include the use of desiccants, opening the 

culture containers, adjusting culture closures or using special closures that 

facilitate water loss. 

2.1.6 Stage IV: In vivo rooting and acclimatization for soil establishment 

DEBERGH and MAENE (1981) listed four problems associated with in vitro 

rooting of regenerated shoots: 

(i) The process of rooting regenerated shoots in vitro is the most labour- 

intensive stage in micropropagation, and has been estimated to be 

responsible for approximately 35 to 75% of the total cost of 

micropropagation in different species; 

(ii) The root system produced in vitro is usually non-functional in a normal 

substrate and thus require formation of new roots after in vivo planting. The 

production of such new roots is accompanied by a cessation of growth; 

(iii) Optimal root formation requires an auxin concentration for the induction 

phase. This auxin concentration could, when present beyond the induction 

phase, inhibit subsequent root elongation; and 

(iv) Damage to the root system during the transplanting process to the soil 

creates openings for the entrance of micro-organisms, resulting in root 

and/or stem diseases. 

19

DEBERGH and MAENE (1981) therefore proposed in vivo rooting of tissue 

cultured shoots as a viable alternative. In vivo rooting is often accomplished either 

by pulse-treating the shoots for some hours, days or weeks with an auxin 

concentration followed by direct planting on normal substrate, dipping the shoots 

in an auxin powder or planting the shoots directly in a rooting mixture previously 

saturated with an auxin solution (DEBERGH and MAENE, 1981; DEBERGH, 

1991). Rooting mixtures include such materials as peat, vermiculite, perlite, bark, 

pumice, rockwool, sand, soil, or their mixtures, with or without supplementation 

with small amount of lime or fertilizer (DEBERGH and MAENE, 1981; TORRES, 

1989). MAENE and DEBERGH (1983) observed that the choice of a substrate, 

the concentration and duration of IBA treatment, and the size of the shoots could 

affect in vivo rooting of regenerated shoots as demonstrated in Cordyline 

terminalis and Ficus benjamina. SHARMA et al. (1999) reported that the 

optimization of harvesting time of microshoots, shoot size, soil pH, plant growth 

regulator treatment, CO2 enrichment and light conditions in specially designed 

hardening chambers significantly affected the success rate of hardening Camellia 

sinensis microshoots. 

The success of micropropagation ultimately lies not only in the production of large 

numbers of plantlets but also on their survival rate in field conditions. As stated by 

RAZDAN (2003), tissue cultured plants generally manifest some structural and 

physiological abnormalities which make them vulnerable to transplantation shocks. 

The author listed such abnormalities to include noticeable decrease in epicuticular 

wax, abnormal leaf morphology and anatomy, poor photosynthetic efficiency, 

malfunctioning of stomata, and hypolignified stems. These abnormalities often 

lead to high water loss, high susceptibility to leaf scorch by sun and poor survival 

of micropropagated plants. SANTAMARIA et al. (1993) however, reported that 

high rates of water loss from micropropagated Delphinium leaves were not due to 

high rates of cuticular transpiration but linked to a lack of stomatal control. In a 

similar vein, SANTAMARIA and KERSTIENS (1994) strongly argued and 

concluded that the lack of control of water loss shown by various micropropagated 

plants is not associated with poor cuticular development, but rather related to the 

failure of stomata to close in response to leaf dehydration. Nevertheless, 

micropropagated plants are often placed in conditions of high relative humidity, 

20

partial shade and good even temperatures for acclimatization before their transfer 

to field conditions (TORRES, 1989). A condition of high relative humidity could be 

created by placing the plants under a transparent plastic tunnel or an intermittent 

mist system (DEBERGH and MAENE, 1981). The relative humidity could then be 

reduced gradually by gradually slitting the plastic bag or reducing the amount of 

mist received by the plants (TORRES, 1989). MURASHIGE (1978) suggested the 

use of foliar feeding until an effective rooting system is developed. According to 

RAZDAN (2003), partial defoliation and the application of transpirants (1% 

Acropol, v/v) in the initial stages of transpiration have been reported to improve the 

survival frequency by reducing water loss. On the other hand, the use of 

antitranspirants to reduce water loss during acclimatization, though suggested as 

a positive measure by some researchers, is said to have mixed results (see the 

review by HAZARIKA, 2003). 

From the above review of the stages in micropropagation, it is clear that success 

in micropropagation depends on a host of factors. The factors could possibly be 

classified into three aspects, which are: (i) the chemical composition and physical 

qualities of the medium (MURASHIGE, 1974), (ii) the culture environment qualities 

(MURASHIGE, 1974), and (iii) the physiology of the plant species. A suitable in 

vitro medium contains all essential nutrients (macro and micro) in appropriate 

proportions, a carbohydrate source (such as sucrose and glucose), vitamins (like 

nicotinic acid and pyridoxine) and plant growth regulators (mainly auxins and 

cytokinins). Some standard basal media formulated and commonly used include 

MS medium (MURASHIGE and SKOOG, 1962), B5 medium (GAMBORG et al., 

1968), White‟s medium (WHITE, 1963), as well as SH medium (SCHENK and 

HILDERBRANDT, 1972). Of these, MS medium remains the most widely used. 

MURASHIGE (1974) observed that the types and concentrations of auxin and 

cytokinin are the most critical organic components of plant propagation media. The 

active but hidden role of tissue endogenous growth regulators in plant growth and 

development must however, not be ignored since the exogenously applied growth 

regulators often interact with the endogenous ones, which may even be of a 

different group (such as abscisic acid, ethylene and gibberellins) (GASPAR et al., 

1996). MURASHIGE (1974) further observed that light and temperature are the 

major environmental factors affecting tissue culture. The effects of auxins and 

21

cytokinins as well as these major environmental factors (light and temperature) on 

in vitro plant regeneration are reviewed in the next two sections of this Chapter. 

2.1.7 Effects of auxins and cytokinins on plant regeneration in micropropagation 

Auxins and cytokinins are usually required for growth and morphogenesis and are 

therefore frequently added to the culture media. According to DAVIES (2004), the 

regulatory role of plant growth regulators in growth and development depends on 

three main factors. These are: (i) the amount present – a factor that is regulated by 

biosynthesis, degradation and conjugation (ii) the location of the growth regulator – 

a factor affected by movement or transport (iii) the sensitivity (or responsiveness) 

of the tissue – a factor which involves the presence of receptors and signal- 

transduction chain components. The exogenous application of many growth 

regulators may however modify the synthesis, degradation, activation, 

sequestration or translocation of, as well as sensitivity to endogenous growth 

regulators of the same or different type (GASPAR et al., 1996; KAMĺNEK et al., 

1997). 

Figure 2.1 presents the structure of some frequently used auxins in plant tissue 

culture. IAA is regarded as the most common naturally occurring auxin in plants 

(MUDAY and DELONG, 2001). In general, auxins exist in plant cells either as free 

acids or in conjugated forms. They can be conjugated to sugars (such as 

monosaccharides and high molecular weight polysaccharides) and sugar alcohols 

(such as myo-inositol) through ester linkages or to single amino acids, peptides 

and proteins through amide linkages (NORMANLY et al., 2004; BAJGUZ and 

PIOTROWSKA, 2009). The formation of auxin conjugates is a mechanism 

involved in the homeostatic control of auxin levels in plant cells (BAJGUZ and 

PIOTROWSKA, 2009). Free auxins, when in excess, can be stored and 

transported in conjugate forms. Such conjugated molecules are protected from 

oxidative breakdown and may be later hydrolysed enzymatically to release free 

auxin when required (GASPAR et al., 1996). The formation and hydrolysis of 

auxin conjugates as well as the conjugate profile of endogenous auxin differ 

among plant tissues and are controlled by the developmental stage of the plant 

(RAMPEY et al., 2004; BAJGUZ and PIOTROWSKA, 2009). The exogenous 

22

application of different auxins may lead to a distinct physiological and/or 

morphogenic response in different explants due to variation in their endogenous 

growth regulator profile at the time of excision, determined by their growth and 

developmental stage. 

Indole-3-acetic acid (IAA) 

Indole-3-butyric acid (IBA) 

α-Naphtalene acetic acid (NAA) 

2,4-Dichlorophenoxyacetic acid (2,4-D) 

Figure 2.1: Molecular structures of some auxins commonly used in plant tissue 

culture. 

23

Auxins are known to play a crucial role in regulating or influencing growth and 

developmental processes such as cell expansion, cell wall acidification, initiation of 

cell division, differentiation of vascular tissue and organization of meristems giving 

rise to either unorganized tissue (callus) or defined organs (GASPAR et al., 1996; 

MUDAY and DELONG, 2001). Responses such as callus production, induction of 

somatic embryogenesis, shoot production, rooting and secondary metabolite 

production in growth media supplemented with different auxin concentrations have 

been reported by many researchers (NIGRA et al., 1987; DE KLERK et al., 1997; 

SUN et al., 2008; AZAD et al., 2009; BASKARAN et al., 2009). Depending on 

the objective, PIERIK (1987) encouraged limiting the use of 2,4–D due to its ability 

to induce mutations and/or inhibit photosynthesis. GASPAR et al. (1996) noted 

that, in addition to the modifying effect of other growth regulators (ALONI, 1995; 

LIU and REID, 1992), the activity of auxins can be affected by the free availability 

of boron. They noted that both the translocation of IAA and nuclear RNA synthesis 

in response to auxin treatment can be inhibited in boron-deficient plants. 

Furthermore, the endogenous level of IAA can be regulated by its photo-oxidation 

(RAY, 1958) when cultures are over-exposed to light. The rapid metabolism of 

auxins such as IAA and IBA within plant tissues may be useful in automatically 

changing the auxin:cytokinin ratio in culture (for example, during callus production 

followed by organogenesis) or when less auxin is required for a subsequent 

developmental phase in the same culture (for example, during root elongation 

following root induction) (GASPAR et al., 1996). 

The structures of some commonly used cytokinins in tissue culture are shown in 

Figure 2.2. Cytokinins such as zeatin and isopentenyladenine with isoprenoid 

derived chains attached at their N 6 terminal are often described as isoprenoid 

cytokinins. The aromatic cytokinins (such as benzyladenine and topolins) have an 

aromatic derivative side chain at their N 6 terminal. Cytokinins can be found in 

different forms such as free bases, ribosides, nucleotides, N-glucosides and O- 

glucosides (OG) in plant cells (LETHAM and PALNI, 1983) and their endogenous 

levels often regulate their physiological activities (KAMĺNEK et al., 1997). The 

exogenous application of cytokinins, their uptake and metabolism in turn influence 

the endogenous cytokinin levels in plant cells (KAMĺNEK et al., 1997). STRNAD 

(1997) classified the metabolism of cytokinins from a physiological perspective into 

24

four main categories. These include: (i) their interconversion, (ii) hydroxylation, (iii) 

conjugation, and (iv) degradation. 

According to LETHAM and PALNI (1983), exogenous cytokinin bases are 

converted by plant tissues into various types of metabolites. Such metabolites 

include products of ring substitution (ribosides, nucleotides, N-glucosides), and 

products of isoprenoid side chain cleavage (adenine, adenosine, adenosine-5'- 

monophosphate) and substitution (O-glucosides) (LETHAM and PALNI, 1983). 

The authors suggested that these metabolites could be active forms of cytokinin, 

translocation forms, storage forms (releasing free cytokinin when needed), or 

detoxification products (formed when exogenous levels become too high and 

toxic), amongst others. According to STRNAD (1997), there is indirect evidence 

suggesting that the free cytokinin bases are most likely the biologically active 

forms of cytokinins. That being the case, the interconversion of cytokinin bases, 

nucleosides and nucleotides is an important process in the regulation of cytokinin 

activity (CHEN, 1997; BAJGUZ and PIOTROWSKA, 2009). The stereo- and/or 

regiospecific hydroxylation of isoprenoid and aromatic cytokinins, respectively, is 

another factor that could regulate cytokinin activity (KAMĺNEK et al., 1979; 

KAMĺNEK et al., 1987a; STRNAD, 1997). KAMĺNEK et al. (1987a) observed that 

the hydroxylation of the phenyl ring of N 6 -benzyladenosine in meta-position 

increased cytokinin activity in tobacco callus and wheat leaf chlorophyll retention 

bioassays. On the other hand, they noted that the hydroxylation of the phenyl ring 

in ortho- and para-positions significantly decreased cytokinin activity, suggesting a 

possible regulation of cytokinin biological activity by a position specific 

hydroxylation of the side chain. 

25

6-Benzyladenine (BA) 

6-Furfurylaminopurine (Kinetin) 

trans-Zeatin (Z) 

N 6 -Isopentenyladenine (iP) 

Dihydrozeatin (DHZ) 

meta-Topolin (mT) 

meta-Topolin riboside (mTR) 

26

meta-Methoxytopolin riboside (MemTR) 

Figure 2.2: Molecular structures of some cytokinins commonly used in plant 

tissue culture. 

STRNAD (1997) noted that the glucosides, unlike the ribosyl and alanyl 

conjugates, are not limited to the N 9 position of the purine ring, hence they appear 

to be the most prominent among the cytokinin conjugates. Owing to this, according 

to the author, cytokinin glycosides are widespread in the plant kingdom and exist 

in different metabolic forms. They play indispensable roles in plant growth and 

developmental processes. In many experiments involving the exogenous 

application of BA, for example, N-glucoside metabolites such as [3G]BA, [7G]BA 

and [9G]BA have been identified in plant cells and tissues, the latter two usually 

being the major ones (LETHAM and PALNI, 1983; WERBROUCK et al., 1995; 

VAN STADEN and CROUCH, 1996). BA is an aromatic cytokinin most widely 

used in the micropropagation industry due to its effectiveness and 

inexpensiveness (BAIRU et al., 2007). The [3G]BA metabolite reportedly exhibited 

a markedly greater cytokinin-like activity in different bioassays compared to 

[7G]BA and [9G]BA, but a weak activity when compared to its corresponding base 

(see the review by VAN STADEN and CROUCH, 1996). As demonstrated in 

radish cotyledons (LETHAM and GOLLNOW, 1985) and soybean callus (VAN 

27

STADEN and DREWES, 1992), the relatively high activity of [3G]BA is likely due 

to cleavage of the 3-glucoside moiety to release free BA (VAN STADEN and 

CROUCH, 1996). On the other hand, the metabolites [7G]BA and [9G]BA are said 

to be products of irreversible inactivation of cytokinins which regulate levels of the 

active free bases (STRNAD, 1997). They are extremely stable in plant tissues and 

biologically relatively inactive, especially [9G]BA (LALOUE, 1977; AUER et al., 

1992). Indeed, the accumulation of [9G]BA in the basal part of Spathiphyllum 

floribundum was reported to be responsible for heterogeneity in growth and 

inhibition of rooting during the acclimatization of this species (WERBROUCK et 

al., 1995). BAIRU et al (2007) were of a similar opinion that the negative effect of 

BA on ex vitro growth of Aloe polyphylla could be due to the accumulation of the 

stable derivative, [9G]BA. 

WERBROUCK et al. (1996) suggested two alternatives to the use of BA in 

micropropagation systems due to the problems associated with its exogenous 

application. One is the use of BA derivatives which are already conjugated at the 

N 9 position (such as [9R]BA) as they are better protected against N 9 -glucosylation. 

The second alternative is the use of hydroxylated BA analogues, mainly the 

topolins. Many researchers have indeed demonstrated the superiority of certain 

topolins over some commonly used cytokinins in micropropagation of different 

species. KUBALÁKOVÁ and STRNAD (1992), for example, observed a high 

shoot formation frequency in sugar beet on medium containing meta-topolin (mT) 

compared to BA and zeatin. WERBROUCK et al. (1996) reported improved 

rooting in Spathiphyllum floribundum with the use of mT compared to BA. The use 

of mT improved shoot multiplication of Curcuma longa compared to BA, kinetin 

and zeatin (SALVI et al., 2002). Other reported effects of topolins include 

improved survival rate of in vitro grown potato (BAROJA-FERNÁNDEZ et al., 

2002), higher multiplication rates of plantain by mT (ESCALONA et al., 2003), 

histogenic stability of Petunia meristem and anti-senescing activity of rose cultures 

by MemTR (BOGAERT et al., 2006), improved multiplication and control of 

hyperhydricity in Aloe polyphylla by mT (BAIRU et al., 2007), superior 

multiplication rates by mT and mTR in two banana cultivars (BAIRU et al., 2008) 

and lower percentages of necrotic shoot-tips in Harpagophytum procumbens in 

treatments containing mTR (BAIRU et al., 2009). 

28

In general, the effectiveness of topolins has been attributed to its distinct 

metabolism from that of BA (STRNAD, 1997). Their metabolites in plant tissues 

are mainly the O-glucosides, a glucosylation made possible by the hydroxy group 

on the benzyl ring (WERBROUCK et al., 1996; MALÁ et al., 2009). STRNAD 

(1997) stated that these metabolites appear to be biologically active intrinsically or 

as products of β-glucosidase activity (LETHAM and PALNI, 1983) and are not 

clear substrates for cytokinin oxidase. Furthermore, studies have revealed that the 

O-glucosides may be storage forms of cytokinin, stable under certain conditions 

and rapidly converted to biologically active forms when required (PARKER et al., 

1978; WERBROUCK et al., 1996; STRNAD, 1997). STRNAD (1997) therefore 

concluded that the reversible sequestration of aromatic cytokinin O-glucosides 

may confer stability and continuous availability over an extended period of time at 

a physiologically active level, resulting in improved shoot multiplication in plants 

cultured in vitro. 

Many developmental processes in plants such as cell growth, cell division and 

differentiation as well as organogenesis in tissue and organ cultures are controlled 

by an interaction between cytokinins and auxins (GASPAR et al., 1996). For 

instance, it is well known that a high cytokinin to auxin ratio generally favours 

shoot production whereas a high auxin to cytokinin ratio favours root formation. It 

should be noted however, that the cytokinin levels could be regulated by auxin 

levels (KAMĺNEK et al., 1997). These authors noted that auxin may down regulate 

cytokinin levels on one hand, by inhibiting cytokinin biosynthesis. On the other 

hand, auxin may promote cytokinin metabolic inactivation by N-glucosylation or 

degradation by cytokinin oxidase. Either way, auxin can strikingly decrease 

cytokinin to auxin ratio and induce certain physiological events (KAMĺNEK et al., 

1997). 

2.1.8 Environmental factors affecting micropropagation 

2.1.8.1 Light 

Light duration (photoperiod), intensity (photosynthetic photon flux, PPF) and 

spectral quality are among the important environmental factors affecting plant 

29

growth and development throughout the culture period (MURASHIGE, 1974). 

They regulate photomorphogenetic processes in tissue cultures and can lead to 

improved plant quality and growth rate (ECONOMOU and READ, 1987). Their 

influence on the levels of endogenous plant growth regulators may elicit different 

morphological responses in different plant species (MACHÁČKOVA et al., 1992; 

TAPINGKAE and TAJI, 2000). KAMADA et al. (1995) reported adventitious bud 

formation in root explants of horseradish treated with auxin alone and cultured 

under a 16 h photoperiod. No adventitious buds were produced when the cultures 

were incubated under continuous darkness. They further observed that treatment 

with cytokinin alone induced bud formation under both continuous dark and 16 h 

light conditions. They attributed their results to light inducing either the activation of 

cytokinin biosynthesis or reduction of cytokinin degradation, resulting in an 

increased endogenous cytokinin level relative to that of auxin. 

Shoot multiplication reportedly improved in shoot-tip cultures of Pelargonium 

species (DEBERGH and MAENE, 1977) and leaf explants of Peperomia 

scandens (KUKLCZANKA et al., 1977) maintained under continuous light. On the 

other hand, ECONOMOU and READ (1986) observed a decrease in the shoot 

length and quality during the first reculture (transfer without subdivision), and in 

number of shoots during the second reculture of azalea (Rhododendron species) 

placed under continuous light. JO et al. (2008) noted that the shoot length, root 

length, number of roots, leaf area, plant fresh weight, dry weight, chlorophyll and 

carotenoid content in Alocasia amazonica increased under shorter day (8/16 h) 

and equinoctial (12/12 h) (light/dark cycle) photoperiods. MORINI et al. (1991) 

however, observed that although shoot production in plum (Prunus cerasifera) was 

not statistically different between 12 and 16 h photoperiods, the 8 h photoperiod 

gave a much lower shoot production rate. In addition, PATERSON and ROSE 

(1979) reported better shoot production in leaf explants of Crassula argenta 

cultured under constant darkness. It is therefore evident that different photoperiods 

may elicit different morphogenic response in different species or explants. 

ECONOMOU and READ (1987), in their review, showed that root formation may 

be inhibited by light duration in some species. They listed some factors likely to be 

responsible for such responses which include insufficient IAA synthesis or IAA 

30

eakdown, photo-inactivation of factors promoting root formation, inhibition of 

rooting cofactor synthesis (phenolic compounds) or stimulation of cofactor 

breakdown as well as production of anatomical or histological barriers. 

JO et al. (2008) observed that the shoot length, bulb size, leaf area, as well as 

fresh and dry matter yields in plants regenerated from Alocasia amazonica leaf 

explants, cultured under very low PPF (15 and 30 µmol m -2 s -1 ) were more or less 

the same. These values however decreased in plants cultured at higher PPF (60 

and 90 µmol m -2 s -1 ). Conversely, ECONOMOU and READ (1986) reported 

increased number, length and quality of shoots obtained from in vitro-derived 

shoot explants of Rhododendron species cultured under PPF of 30 and 75 µmol 

m -2 s -1 compared to those cultured under a 10 µmol m -2 s -1 PPF. In a similar vein, 

TAPINGKAE and TAJI (2000) recorded a significant increase in the dry weight of 

Anigozanthos bicolor roots with an increase in PPF (up to 200 µmol m -2 s -1 ). They 

further observed the highest number of roots under a white light while yellow and 

red lights significantly increased root length, suggesting the participation of the 

phytochrome system in the rhizogenesis of this species. The authors were of the 

opinion that the rate of auxin breakdown in the media over time may be faster 

under red and yellow light conditions, resulting in increased root elongation. 

2.1.8.2 Temperature 

In order to derive the most benefits from the tissue culture propagation of a 

particular plant species, its precise temperature needs must be met 

(MURASHIGE, 1974). This is particularly important as the activities of the 

enzymes involved in many biochemical reactions increase at optimum 

temperatures. On the other hand, low temperature could modify the levels of plant 

growth regulators by stimulating the activity of cytokinin oxidase (VESELOVA et 

al., 2005), prolong cell cycle process and activate cold acclimation signalling 

pathways (XIA et al., 2009). In general, in vitro cultures are often maintained at a 

constant temperature of approximately 25°C in many micropropagation protocols. 

In an experiment carried out on four accessions of mint plants (Mentha spp.), 

ISLAM et al. (2005) observed that both the apical and nodal explants cultured at 

25°C exhibited better in vitro growth compared to those cultured at 20°C. 

31

HASEGAWA et al (1973) reported a constant temperature of 27°C as the 

optimum temperature for in vitro plant formation of Asparagus officinalis through 

shoot apex culture. Although many annual and tropical species whose life cycles 

are completed during a period of relatively uniform temperature conditions may 

respond well under constant temperatures, some other species such as those 

adapted to temperate and desert climates may respond better under periodically 

varied temperature conditions (MURASHIGE, 1974). KOZAI et al (1995) reported 

increases in stem length and internode length of Solanum tuberosum cultures as 

the difference in photoperiod and dark period temperatures (DIF) increased. An 

increase in plantlet growth with increased DIF was similarly reported in Rehmannia 

glutinosa (CUI et al., 2001). 

2.1.9 Tissue culture of the families: Acanthaceae and Asclepiadaceae 

With the exception of published articles from this research, the available literature 

reveals no published reports on the in vitro propagation of Barleria species and 

Huernia species. However, successful micropropagation has been reported in the 

families Acanthaceae and Asclepiadaceae (Table 2.1), to which the genera 

Barleria and Huernia belong. As Table 2.1 shows, different parts of a plant can 

serve as explant sources and regeneration can be accomplished through axillary 

shoot proliferation, adventitious shoot production and/or somatic embryogenesis. 

In addition, the production of valuable secondary metabolites was reported in 

induced callus in some cases. 

32

Table 2.1: List of some tissue cultured plant species in the Acanthaceae and Asclepiadaceae families 

Plant species Explant Response Reference 

Acanthaceae 

Adhatoda beddomei Node Axillary shoots, callus SUDHA and SEENI (1994) 

Adhatoda vasica Shoot-tip Axillary shoots ANAND and BANSAL (1998) 

Adhatoda zeylanica Leaf, petiole Callus, somatic embryos, roots JAYAPAUL et al. (2005) 

Andrographis paniculata Leaf, internode Callus, somatic embryos MARTIN (2004a) 

Aphelandra sinclairiana Anther, root, leaf Callus NEZBEDOVÁ et al. (1999) 

Aphelandra tetragona Petiole, leaf Callus NEZBEDOVÁ et al. (1999) 

Crossandra infundibuliformis Bud Axillary shoots GIRIJA et al. (1999) 

Justicia gendarussa Leaf, node Callus, adventitious shoots RAGHUVANSHI et al. (1994), AGASTIAN et 

Nilgirianthus ciliatus Leaf, node, 

Asclepiadaceae 

internode 

al. (2006) 

Callus, adventitious shoots DEVI and KAMALAM (2007) 

Asclepias curassavica Node Shoots, roots, callus PRAMANIK and DATTA (1986) 

Calotropis gigantean Hypocotyl, 

immature embryo, 

stem, leaf 

Callus, adventitious shoots ROY et al. (2000), ROY and DE (1990) 

Calotropis procera Cotyledon Callus, laticifer differentiation SURI and RAMAWAT (1996) 

33

Table 2.1 continued 


Ceropegia bulbosa 

var. bulbosa 

Ceropegia bulbosa 

var. lushii 

Node, stem, leaf, 

bud 

Ceropegia candelabrum Node, leaf, 

Axillary shoots, adventitious shoots, 

callus, flowering, microtubers, 

somatic embryos 

Node, stem, leaf Axillary shoots, callus, microtubers, 

internode 

somatic embryos 

Axillary shoots, callus, somatic 

embryos 

Ceropegia hirsuta Bud Axillary shoots, adventitious shoots, 

flowering 

Ceropegia jainii Node, stem, leaf Axillary shoots, callus, microtubers, 

somatic embryos, flowering 

Ceropegia juncea Node, internode Axillary shoots, callus, adventitious 

shots, roots 

Ceropegia lawii Bud Axillary shoots, adventitious shoots, 

flowering 

Ceropegia maccannii Bud Axillary shoots, adventitious shoots, 

flowering 

Ceropegia oculata Bud Axillary shoots, adventitious shoots, 

flowering 

Ceropegia sahyadrica Bud Axillary shoots, adventitious shoots, 

flowering 

PATIL (1998), NAIR et al. (2007), BRITTO et 

al. (2003) 

PATIL (1998) 

BEENA et al. (2003), BEENA and MARTIN 

(2003) 

NAIR et al. (2007) 

PATIL (1998) 

NIKAM and SAVANT (2009) 





34

Table 2.1 continued 


Cryptolepis buchanani Leaf, internode Callus VENKATESWARA et al. (1987) 

Decalepis hamiltonii Leaf, shoot-tip Callus, somatic embryos, 

Gymnema sylvestre Node, hypocotyl, 

cotyledon, leaf 

Hemidesmus indicus Leaf, root, node, 

shoot-tip, root 

Holostemma ada-kodien Node, internode, 

leaf 

adventitious shoots 

Axillary shoots, callus,somatic 

embryos 


embryogenesis, adventitious shoots 

Axillary shoots, callus, adventitious 

shoots, somatic embryos 

GIRIDHAR et al. (2004, 2005) 

KOMALAVALLI and RAO (2000), ASHOK 

KUMAR et al. (2002) 

PATNAIK and DEBATA (1996), SARASAN 

et al. (1994), MISRA et al. (2005), RAMULU 

et al. (2003) 

MARTIN (2002), MARTIN (2003) 

Holostemma annulare Shoot-tip, node Axillary shoots, callus SUDHA et al. (1998) 

Leptadenia reticulata Node, leaf, 

internode, shoot tip 


embryos 

ARYA et al. (2003), MARTIN (2004b) 

Pergularia daemia Shoot-tip, node Shoots KIRANMAI et al. (2007) 

Sarcostemma brevistigma Node Axillary shoots, callus, adventitious 

Tylophora indica Stem, leaf, node, 

petiole 

shoots 

Callus, somatic embryos, axillary 

shoots, adventitious shoots 

THOMAS and SHANKAR (2009) 

RAO and NARAYANASWAMI (1972), 

MANJULA et al. (2000), SHARMA and 

CHANDEL (1992), THOMAS and PHILIP 

(2005), FAISAL et al. (2005) 

35

2.2 Pharmacological and phytochemical investigation of plant extracts 

2.2.1 Introduction 

The use of natural products in disease prevention and control as well as in drug 

development has received increased attention in recent times. According to 

RATES (2001), about 25% of the globally prescribed drugs are obtained from 

plants. The author observed that 11% of the 252 drugs considered as basic and 

essential by the World Health Organisation are solely of plant origin. This growing 

interest in the therapeutic use of drugs of plant origin has been attributed to some 

factors such as (i) the side effects and ineffective therapy of conventional 

medicine, (ii) the incorrect and/or abusive use of synthetic drugs resulting in 

undesired side effects and other problems, (iii) the inaccessibility of a large part of 

the world‟s population to conventional pharmacological treatment, and (iv) the 

suggestion created by folk medicine and ecological awareness that natural 

products are often harmless (RATES, 2001). In many parts of the world, especially 

in Africa, the increasing use of plants in traditional medicines by a growing 

population, among other reasons, has resulted in the over-exploitation of many of 

our plant resources. 

Plants, in addition to their therapeutic use in herbal preparations, can serve as 

important sources of new drugs, new drug leads and new chemical entities 

(BALUNAS and KINGHORN, 2005). However, a large percentage of the 

estimated 350, 000 plant species on earth is yet to be investigated for their 

pharmacological and phytochemical potential (HOSTETTMAN and MARSTON, 

2002). SHAI et al. (2008) for example, noted that some South African indigenous 

plant species are at risk of becoming extinct before the investigation and 

application of their potential as sources of therapeutic drugs can be carried out. 

HOSTETTMANN and MARSTON (2002) recommended that urgent attention be 

given to the exploration of this „green inheritance‟ for their pharmacological and 

phytochemical potential, especially due to the rapid disappearance of our tropical 

forests and other important vegetation areas. Some of these plant resources 

(including those rapidly disappearing) could contain novel, active chemotypes that 

can serve as leads for effective drug development (CRAGG et al., 1997). 

36

The dual chemical-biological screening approach has been recommended as the 

fastest method of discovering important plant-derived bioactive compounds 

(HOSTETTMAN and MARSTON, 2002). The localisation of such active 

compounds however, requires the use of simple but sensitive target-specific 

bioassays, since plant extract often contains low concentrations of active 

compounds (RATES, 2001; HOSTETTMAN and MARSTON, 2002). The main 

targets for biological tests can be lower organisms (bacteria, fungi and viruses), 

invertebrates (insects, crustaceans and molluscs), isolated subcellular systems 

(enzymes and receptors), animal or human cell cultures, isolated organs of 

vertebrates, or whole animals (HOSTETTMAN and MARSTON, 2002). 

Furthermore, since plants often contain a large number of promising compounds, 

there is a need to use a variety of test systems in order to detect other potential 

bioactivities (RATES, 2001; HOUGHTON et al., 2007). The use of solvents of 

increasing polarity during extraction processes has also been recommended, 

especially when the chemical composition is unknown (WILLIAMSON et al., 1996; 

cited by RATES, 2001). 

2.2.2 Antimicrobial activity 

Some bacteria and fungi have been implicated in the pathology of many diseases. 

For example, the fungus Candida albicans is known to be a pathogenic organism 

causing candidosis (SHAI et al., 2008), while bacteria such as Eschericha coli, 

Staphylococcus aureus and Klebsiella pneumoniae are known to be involved in 

gastroenteritis or respiratory infections (MOISE and SCHENTAG, 2000; SUN et 

al., 2006). The occurrence of new and re-emerging infectious diseases with no 

effective therapies as well as the development of resistant pathogen strains to 

some currently used drugs necessitate the urgent and continuous search for 

potent and efficacious antimicrobial compounds (CRAGG et al., 1997). 

In the biological screening of plants for antibacterial activity, COS et al. (2006) 

recommended the use of a panel of test organisms consisting of, at least, a Gram- 

positive (such as S. aureus and Bacillus subtilis) and a Gram-negative (such as E. 

coli and K. pneumonia) bacterium. The use of yeasts (such as C. albicans), 

dermatophytes (such as Trichophyton mentagrophytes) and opportunistic 

37

filamentous fungi (such as Fusarium solani), are among the recommended fungi in 

the screening for antifungal activity (COS et al., 2006). These authors further 

suggested the use of American type culture collection (ATCC) strains as they are 

well-characterized. 

Besides the choice of test organisms, of vital importance is the choice of an 

appropriate screening method. The three commonly used antimicrobial screening 

methods are (i) agar-diffusion, (ii) bio-autographic, and (iii) dilution assays (COS et 

al., 2006; VAN VUUREN, 2008). The agar-diffusion method is said to be simple 

and can be used to screen a large number of test samples against one micro- 

organism (RĺOS and RECIO, 2005; VAN VUUREN, 2008). However, the 

differences in physical properties of test samples, such as the solubility, volatility 

and diffusion characteristics in agar, make it difficult to effectively compare the 

antimicrobial activity of different samples using the agar-diffusion method (COS et 

al., 2006). The bio-autographic assay, although highly sensitive, requires the 

complete removal of residual low volatile solvents and is applicable mainly to 

micro-organisms that easily grow on thin-layer-chromatographic (TLC) plates 

(COS et al., 2006). It is therefore mainly used in the isolation of bioactive 

antimicrobial compounds (VAN VUUREN, 2008). The dilution assay allows for the 

quantitative determination of the minimum inhibitory concentrations (MIC) for 

comparing the antimicrobial potency of test samples (VAN VUUREN, 2008). In 

addition to its usefulness in assaying polar and non-polar extracts as well as 

isolated compounds, the dilution assay method can be used to determine whether 

an extract or a compound has a lethal (-cidal) or static action at a particular 

concentration (COS et al., 2006). In general, as benchmarks for determining the 

potency of extracts, extracts with MIC values below 8 g/ml are considered as 

having some antimicrobial activity (FABRY et al., 1998) while those with MIC 

values less than 1 mg/ml are regarded as exhibiting interesting activity (RĺOS and 

RECIO, 2005). 

Antimicrobial activity has been reported in some Barleria species. For example, 

MAREGESI et al. (2008) reported a MIC value of 1 mg/ml in B. eranthemoides 

root methanolic extract tested against Bacillus cereus. CHOMNAWANG et al. 

(2009) reported MIC values of 5.0, 2.5 and 5 mg/ml in B. lupulina ethanolic extract 

38

tested against S. aureus, S. epidermidis and methicillin-resistant S. aureus, 

respectively. Unspecified extracts of B. lupulina were reported to have minimum 

bactericidal concentration (MBC) values of 1.25 and 5.0 mg/ml against 

Propionibacterium acnes and S. epidermidis, respectively (CHOMNAWANG et al., 

2005). KOSMULALAGE et al. (2007) reported antibacterial activity of crude 

ethanolic extract of the aerial part of B. prionitis against S. aureus and 

Pseudomonas aeruginosa using the paper disk diffusion method. The authors 

further reported the antibacterial activity of three compounds isolated from this 

ethanolic extract against Bacillus cereus and P. aeruginosa (25 μg/disk) using the 

same method. As far as can be ascertained from the available literature however, 

no report has been made (with the exception of published materials from this 

thesis) on the antibacterial and antifungal activities of the different parts of the 

three Barleria species in the present study as well as of any Huernia species. 

2.2.3 Anti-inflammatory activity 

Inflammation is a process implicated in the pathogenesis of many diseases like 

Alzheimer‟s disease, asthma and auto-immune diseases such as rheumatoid 

arthritis, multiple sclerosis and ulcerative colitis (HOWES and HOUGHTON, 2003; 

POLYA, 2003). It is an immunological response often elicited by tissue injury from 

bacterial and viral infection, wounding and other sources of damage (POLYA, 

2003; BYEON et al., 2008). Currently, nonsteroidal anti-inflammatory drugs 

(NSAIDs) are commonly used in the symptomatic treatment of inflammation (VIJI 

and HELEN, 2008). In fact, NSAIDs are reported to be among the most widely 

used drugs worldwide (FIORUCCI et al., 2001; STEINMEYER and KONTTINEN, 

2006). Nevertheless, the unwanted side effects such as gastric intolerance, water 

and electrolyte retention, bone marrow depression and cardiovascular risks 

associated with the use of these drugs (XIAO et al., 2005; STEINMEYER and 

KONTTINEN, 2006) have necessitated further search for „classical‟ anti- 

inflammatory drugs with reduced or no side effects. 

Inflammation is a complex process involving the release of highly active pro- 

inflammatory mediators such as prostaglandins, leukotrienes, histamine, cytokines 

and free radicals (ZSCHOCKE et al., 2000a). Prostaglandins, such as PGE2, 

39

PGI2, PGF2α, are known to sensitize primary afferent nociceptive nerve fibers, 

which contributes to inflammatory pain (KONTTINEN et al., 1994). The release of 

prostaglandins from arachidonic acid is catalysed by the action of the 

cyclooxygenase (COX) enzyme (also known as prostaglandin-H2-synthase, PGH2 

synthase) (STEINMEYER, 2000). The bi-functional COX enzyme catalyses the 

oxygenation of arachidonic acid (hydrolytically released from membrane 

phospholipids) to form the cyclic prostaglandin endoperoxide PGG2, followed by a 

peroxidase reaction which results in the formation of PGH2 (FRÖLICH, 1997; 

STEINMEYER, 2000). PGH2 is the precursor of other bioactive prostaglandins and 

thromboxanes (STEINMEYER, 2000). NSAIDs are known to inhibit the production 

of prostaglandins mainly by inhibiting COX activity (VANE, 1994). 

The COX enzyme is known to exist mainly in two isoforms (COX-1 and COX-2), 

although the possible existence of a third isoform (COX-3) has been hypothesized 

(BERENBAUM, 2004). The COX-1 and COX-2 isoenzymes are said to have 

approximately 60% homology in their nucleic acid and amino acid structures 

(VANE, 1994). COX-1 is identified to be constitutively expressed in most tissues 

with prostaglandin synthesis for maintaining some physiological functions such as 

gastric mucosa protection and renal perfusion maintenance (LI et al., 2006). On 

the other hand, COX-2 was thought to be induced only under pathological 

conditions and thus is solely responsible for the production of inflammatory 

prostanoid mediators (STEINMEYER and KONTTINEN, 2006). This concept led 

many researchers to start focussing on selective COX-2 inhibitors. However, 

recent published studies have shown that COX-2 is also constitutively expressed 

in some body parts such as the brain, spinal cord, kidney and uterus 

(STEINMEYER and KONTTINEN, 2006). As illustrated in Figure 2.3, COX-2 

isoform has both physiological and pathophysiological roles. Furthermore, the use 

of some selective COX-2 inhibitors have been reported to carry a risk of gastro- 

intestinal toxicity in some patients (MACAULAY and BLACKBURN, 2002; 

BERTIN, 2004; WARNER and MITCHELL, 2008). STEINMEYER and 

KONTTINEN (2006) further stated some undesirable side effects such as water 

and electrolyte retention, delayed wound healing (in animal experiments) and 

cardiovascular risks reported with the use of selective COX-2 inhibitors. According 

to these authors, several selective COX-2 inhibitors have already been withdrawn 

40

from the market due to their cardiovascular risk and at present, all COX-2 selective 

NSAIDs are surmised to be likely causing heart attacks, strokes and thromboses. 

They therefore recommended the classical combination of a non-selective NSAID 

with a proton pump inhibitor (such as omeprazol) or misoprostol as a viable, low- 

cost alternative to COX-2 selective NSAIDs in the prophylaxis of the NSAID- 

gastropathy. 

On the other hand, the inhibition of COX enzyme is known to up-regulate the 

production of leukotrienes (LTs) from arachidonic acid through the 5-lipoxygenase 

(LOX) enzyme pathway (FIORUCCI et al., 2001). Leukotrienes are involved in the 

pathophysiology of chronic inflammation and allergic diseases such as rheumatoid 

arthritis, asthma as well as skin diseases like psoriasis (ZSCHOCKE et al., 

2000a). The leukotriene LTB4, in particular, has been reported to have a 

pathophysiological function in the development of gastrointestinal ulcers (ASAKO 

et al., 1992; cited by FIORUCCI et al., 2001). Many authors have therefore 

suggested that the use of compounds that are dual inhibitors of COX and LOX 

enzymes could enhance anti-inflammatory effects with reduced undesirable side 

effects (ZSCHOCKE et al., 2000a; FIORUCCI et al., 2001; LI et al., 2006; VIJI 

and HELEN, 2008). These authors were of the opinion that dual inhibitors of COX 

and LOX enzymes could be a potential viable alternative to standard NSAIDs and 

selective COX-2 inhibitors. 

Figure 2.3: The physiological and pathophysiological functions of COX-2 

enzyme (STEINMEYER, 2000). 

41

Many researchers have reported anti-inflammatory activity in plant extracts using 

different in vivo animal models in their evaluation. SINGH et al. (2003) 

demonstrated the anti-inflammatory activity of the n-butanol and aqueous fractions 

of the whole B. prionitis plant against carrageenan-induced oedema in rats. 

WANIKIAT et al. (2008) reported the anti-inflammatory effect of B. lupulina 

methanolic extract in carrageenan-induced paw oedema and ethyl 

phenylpropiolate-induced ear oedema in rats. However, according to TALHOUK 

et al. (2007), studies reporting anti-inflammatory activities of crude plant extracts 

in whole animal models demonstrate gross anti-inflammatory activities but typically 

fall short of identifying possible intracellular targets. Some of the common key 

molecules at the cellular level in the inflammatory cascade that are targeted by 

plant extracts include nuclear factor-κB, cytokines and COX (TALHOUK et al., 

2007). Assays involving enzymes are generally known to be highly specific, very 

sensitive and mechanism-based, and they are valuable in screening large 

numbers of samples as they are relatively easy and small amounts of material are 

required (HOSTETTMANN and MARSTON, 2002). 

2.2.4 Acetylcholinesterase inhibition 

Acetylcholinesterase (AChE) is an enzyme involved in the hydrolysis of 

acetylcholine into a choline and acetyl group (DOHI et al., 2009). Acetylcholine is 

a neurotransmitter at all preganglionic autonomic, parasympathetic and some 

sympathetic postganglionic nerve endings, neuromuscular junctions and at some 

central nervous system (CNS) synapses (KOSMULALAGE et al., 2007). 

Neurotransmitter disturbances and low cholinergic function are among the 

pathological features involved in CNS disorders (HOWES and HOUGHTON, 

2003). The inhibition of the AChE enzyme, leading to the maintenance of 

acetylcholine level and enhanced cholinergic function has become the standard 

approach in the symptomatic treatment of Alzheimer‟s disease (AD) (HOWES and 

HOUGHTON, 2003; VINUTHA et al., 2007). AD is the most common type of 

neurodegenerative disease, characterized primarily by impaired memory and 

cognitive dysfunction, and at advanced stages, language deficit, depression, 

agitation, mood disturbances and psychosis (HOUGHTON et al., 2007). The 

undesirable side effects such as hepatotoxicity in the therapeutic use of some 

42

AChE inhibitors (like tacrine) coupled with their limitation to symptom alleviation 

has necessitated further search for more potent drugs (LÓPEZ et al., 2002; 

HOWES and HOUGHTON, 2003; FERREIRA et al., 2006). 

KOSMULALAGE et al. (2007) recently reported moderate AChE inhibitory 

activities of four compounds isolated from ethanolic extract of B. prionitis. These 

compounds were identified to be balarenone, pipataline, lupeol and 13,14-seco- 

stigmasta-5,14-diene-3-α-ol. They also reported a significant AChE inhibitory 

activity with an IC50 value of 36.8 µM in the compound 8-amino-7- 

hydroxypipataline, a synthetic derivative of pipataline. ATA et al. (2009) reported 

the isolation of a new phenylethanoid glycoside (barlerinoside) along with six 

known iridoid glycosides (shanzhiside methyl ester, 6-O-trans-p-coumaroyl-8-O- 

acetylshanzhiside methyl ester, barlerin, acetylbarlerin, 7-methoxydiderroside and 

lupulinoside) from the ethanolic extract of B. prionitis aerial parts. These 

compounds were reported to exhibit weak AChE inhibitory activities with IC50 

values of 175.9, 133.5, 150.0, 100.0, 145.8, 190.0 and 178.9 µM, respectively. The 

possibility of a synergistic AChE inhibition of two or more of these compounds 

cannot be completely ruled out. Synergistic interaction was reported to be 

responsible for stronger AChE inhibition in Salvia lavandulaefolia essential oil 

compared with that of its constituent terpenes (SAVELEV et al., 2003). The 

authors also reported both synergistic and antagonistic responses in some 

combinations of this plant terpene constituent. Taken together however, the 

reported results on B. prionitis perhaps suggest the possibility of discovering 

potential effective AChE inhibitors or leads from Barleria species. At present, there 

is no report available on the AChE inhibitory activities of the individual plant parts 

of B. prionitis and other Barleria species. 

2.2.5 Antioxidant activity 

Free radical reactions have been implicated in the pathology of a large number of 

disease states such as cancer, AD, diabetes, inflammation and several 

cardiovascular diseases (HOUGHTON et al., 2007). According to HOUGHTON 

and HOWES (2003), the use of antioxidants has been shown to slow AD 

progression and neuronal degeneration. HOUGHTON et al. (2007) suggested the 

43

inclusion of antioxidant and free radical scavenging tests, among others, in the 

biological screening of plants used for wound healing. This is due to the fact that 

the wound healing process is impaired by microbial infection and destruction of 

cells and tissues by reactive oxygen species (ROS) (HOUGHTON et al., 2007; 

PATTANAYAK and SUNITA, 2008). ROS can damage cellular components and 

act as secondary messengers in the inflammation process (FERREIRA et al., 

2006). Antioxidants however, have the ability to scavenge ROS, attenuate 

inflammation pathways, reduce cancer, heart disease and other degenerative 

problems associated with aging (HENRY et al., 2002; FERREIRA et al., 2006). 

The realization of antioxidant roles in the management of some disease states has 

led to the inclusion of antioxidant tests in many pharmacological screenings of 

plant extracts and isolated compounds. According to HUANG et al. (2005), the 

major antioxidant assays can be divided into two categories, based on the 

chemical reactions involved. These two categories are (i) hydrogen atom transfer 

(HAT) reaction based assays and (ii) single electron transfer (ET) reaction based 

assays. In the HAT based assays, a synthetic free radical generator, an oxidizable 

molecular probe and an antioxidant are generally involved, and the assays 

measure the competitive kinetics of the reaction (HUANG et al., 2005). The ET 

based assays measure the antioxidant capacity of an extract or a compound in the 

reduction of an oxidant, a probe indicating the reaction endpoint (HUANG et al., 

2005). Some examples of HAT based assays include inhibition of linoleic acid 

oxidation, ORAC (oxygen radical absorbance capacity) and Crocin bleaching 

assays while the ET based assays include FRAP (ferric ion reducing power 

assay), DPPH (1,1-diphenyl-2-picrylhydrazyl) and TEAC (Trolox equivalent 

antioxidant capacity) assays (HUANG et al., 2005). The use of the DPPH assay in 

antioxidant screening is very common due to its simplicity and reproducibility 

(KATSUBE et al., 2004). Nevertheless, due to the fact that antioxidant processes 

are complex, the use of at least two different types of assays in antioxidant activity 

study has been recommended (MOON and SHIBAMOTO, 2009). These authors 

recommended the combination of assays for scavenging electrons or radicals with 

assays associated with lipid peroxidations. 

44

2.2.6 Phytochemical property 

Plants often contain secondary metabolites, many of which are known to 

demonstrate good biological activity in pharmacological screening. The common 

groups of secondary metabolites in plants include the phenolics, terpenes and 

alkaloids. Phenolic compounds are a class of secondary metabolites generally 

having an aromatic ring with one or more hydroxyl substituents (ROBARDS et al., 

1999). ROBARDS et al. (1999) defined phenolics based on their metabolic origin 

as substances derived from the shikimate pathway and phenylpropanoid 

metabolism. They are extremely diverse both in their structures and functions 

(POLYA et al., 2003). The presence of -OH groups in the phenolic ring system 

allows for the polarity and water-solubility of phenolic compounds as well as their 

capacity for hydrogen bonding (POLYA, 2003). The capacity of the phenolic group 

to be deprotonated and oxidised explains their biologically important antioxidant 

properties (POLYA, 2003). Through covalent reaction with free radicals, especially 

the ROS, many phenolics can serve as good anti-inflammatory and antioxidant 

agents (POLYA, 2003). Some common phenolics in plants include tannins 

(hydrolysable tannins and condensed tannins) and flavonoids (the most 

widespread and diverse of the phenolics) (ROBARDS et al., 1999; POLYA, 

2003). 

Tannins are widely distributed defensive compounds in plants (POLYA, 2003). 

According to NIEMETZ and GROSS (2005), they are commonly classified into two 

categories: (i) condensed tannins (also known as proanthocyanidins), and (ii) 

hydrolysable tannins. Condensed tannins are essentially of flavonoid origin while 

hydrolysable tannins are further divided into two subclasses: (i) gallotannins, which 

involve a glucose esterified to gallic acid, and (ii) ellagitannins, which are ellagic 

acid-derived hexahydroxydiphenic acid (POLYA, 2003). The distribution of 

gallotannins is rather limited in nature while ellagitannins are commonly found in 

many plant families (NIEMETZ and GROSS, 2005). OKUDA (2005) stated that 

the profile of hydrolysable tannin in a plant species is generally stable throughout 

the year. AKIYAMA et al. (2001) reported the antibacterial activity of several 

tannins against 18 strains of S. aureus, including 11 strains that are methicillin- 

resistant. ENGELS et al. (2009) and TIAN et al. (2009a, b) observed antimicrobial 

45

activity of gallotannins from Mangifera indica and Galla chinensis, respectively. 

The antimicrobial activity of hydrolysable tannins have been linked to (i) their 

ability to interact with proteins and inhibit enzyme activities, (ii) the damage of lipid 

bilayer membranes as demonstrated in Helicobacter pylori, and (iii) their ability to 

complex metal ions (such as iron) (ENGELS et al., 2009). Furthermore, OKUDA 

(2005) stated that tannins are generally not mutagenic (based on Ames‟ test), but 

instead, they showed antimutagenic activity against certain mutagens. Their 

radical scavenging ability as well as the inhibition of lipid peroxidation and enzyme 

activities (such as LOX) are said to be the activities underlying the antioxidant and 

anti-inflammatory effects of tannins (OKUDA, 2005). 

Flavonoids are well characterized in plant extracts as the main compounds 

possessing anti-inflammatory activity (TALHOUK et al., 2007). They inhibit a 

variety of molecules such as nuclear factor-кB (NF-кB), inducible nitric oxide 

synthase (iNOS), cytokines, COX, LOX and matrix metalloproteinases (TALHOUK 

et al., 2007). CHI et al. (2001) reported various degrees of inhibition of COX-1, 

COX-2, 5-LOX and/or 12-LOX by 19 naturally occurring flavonoids isolated from 

medicinal plants. TUNALIER et al. (2007) reported both antioxidant and anti- 

inflammatory activities in flavonoid rich polar extracts of Lythrum salicaria. 

PATTANAYAK and SUNITA (2008) observed antibacterial and antifungal 

activities of Dendrophthoe falcata flavonoid-containing extracts, using 12 and 5 

different bacterial and fungal strains, respectively. They concluded that the marked 

activity recorded was due to the presence of flavonoids and terpenes in the 

extracts. 

Many authors have reported the isolation of iridoids in some Barleria species. ATA 

et al. (2009) reported the isolation of seven iridoids from the aerial parts of B. 

prionitis, with different levels of biological activity in glutathione S-transferase 

(GST) inhibition, AChE inhibition and free radical scavenging assays. CHEN et al. 

(1998) reported the isolation of some iridoids from B. prionitis with antiviral potency 

against the respiratory syncytial virus. SINGH et al. (2004) observed a significant 

and concentration-dependent hepatoprotective potential of the iridoid enriched 

fraction from B. prionitis extract. The isolation and characterization of iridoids have 

also been reported in other Barleria species such as B. strigosa 

46

(KANCHANAPOOM et al., 2004) and B. lupulina (KANCHANAPOOM et al., 

2001). The anti-inflammatory activity of some plant extracts has been attributed to 

the presence of iridoids (LEVIEILLE and WILSON, 2002). BOLZANI et al. (1997) 

have reported the antifungal property of certain iridoids such as gardiols. Iridoids 

generally are monoterpenes deriving biosynthetically from geranylpyrophosphate 

and can be found in free or glycoside form in plant extracts (POLYA, 2003). 

47

3.1 Introduction 

Chapter 3 In vitro propagation of Barleria greenii 

The application of tissue culture techniques in the propagation of ornamental 

plants has recorded a big stride in recent years and has become widely accepted 

as a standard practice in the horticultural industry (READ and PREECE, 2009). At 

present, more than one billion traded ornamental plants are reportedly produced 

through tissue culture (PRAKASH, 2009). A large percentage of this figure 

focuses on cut flowers and pot plants whereas ornamental perennials and garden 

plants account for only 2% of the total ornamental plants produced through tissue 

culture (PRAKASH, 2009). It has been estimated that the global trade of 

ornamental perennials alone is about 8 billion US dollars per year compared to 90 

and 60 billion US dollars for cut flower and pot plants, respectively (PRAKASH, 

2009). There is indeed a great need as well as a great potential for the application 

of tissue culture techniques in propagating ornamental perennials and garden 

plants. 

The major challenges or setback in the application of tissue culture techniques to 

ornamental perennials and garden plants include the recalcitrant nature of plant 

tissues, often related to seasonal dormancy, establishment of clean explants, slow 

multiplication rate, poor rooting frequency, morphological aberrations and high 

cost of production (PRAKASH, 2009). The choice of plant growth regulators has 

however been reported to play a crucial role in solving or alleviating most of these 

problems (WERBROUCK et al., 1995, 1996; BAIRU et al., 2007, 2008, 2009). 

The use of topolins, for example, has been reported to increase shoot 

multiplication, maintain histogenic stability, improve rooting efficiency and 

subsequently reduce the production cost (KUBALÁKOVÁ and STRNAD, 1992; 

WERBROUCK et al., 1996; BOGAERT et al., 2006). It is therefore important that 

the choice of appropriate plant growth regulators, amongst others, be given close 

attention when developing micropropagation protocols especially suitable for 

commercial application. 

48

Barleria greenii (Family: Acanthaceae) is a perennial ornamental shrub, endemic 

to a specific area in the KwaZulu-Natal province, South Africa. SCOTT-SHAW 

(1999) described it as a successful garden plant with horticultural potential. This 

plant species was discovered in 1984 (BALKWILL et al., 1990) and is known to 

be found only in eight localities on three farms near Estcourt, South Africa 

(MAKHOLELA et al., 2003). It is currently listed in the National Red List of South 

African plants as critically endangered (SANBI, 2009). Barleria greenii can be 

grown from seed as well as from cuttings. The propagation from seed is often 

affected by low seed viability as a result of high seed parasitism. MAKHOLELA et 

al. (2003) observed that in instances where there might have been successful 

pollination and seed production, survival of the seed into the next generation is 

often prohibited by seed parasites, resulting in a highly reduced number of viable 

seeds. SCOTT-SHAW (1999) therefore suggested that where viable seeds are not 

available, propagation by cuttings, although difficult, is the best option. 

This study was aimed at developing an effective micropropagation protocol, which 

can potentially provide a conservation measure for this endangered plant species. 

The effects of different cytokinins and photoperiods were also investigated in this 

study. As far as can be ascertained from the available literatures, there is no report 

to date on micropropagation of any Barleria species. 

3.2 Materials and methods 

3.2.1 Explant decontamination, selection and bulking 

Shoot-tip and nodal explants were excised from stock plants (Figure 3.1A) 

maintained in the shade house in the University of KwaZulu-Natal Botanical 

Garden. The explants were thoroughly rinsed under running tap water before they 

were subjected to surface decontamination treatments. The treatments involved 

soaking the plant materials in 70% ethanol for 60 sec followed by 3.0% sodium 

hypochlorite solution for 20 or 30 min, or 3.5% for 20 min. In each solution, a few 

drops of Tween 20 were added as a surfactant. The surface decontaminated 

explants were cut into 10 mm lengths and cultured individually on 10 ml (in culture 

tubes, 100 mm × 25 mm, 40 ml volume) of full strength MS medium supplemented 

49

with 30 g l -1 sucrose, 0.1 g l -1 myo-inositol, 3.0 µM BA and solidified with 8 g l -1 

agar (Bacteriological agar–Oxoid Ltd., Basingstoke, Hampshire, England). The pH 

of the medium was adjusted to 5.7 with KOH or HCl before autoclaving at 121 o C 

and 103 kPa for 20 min. Cultures were incubated in a growth room with 16 h 

photoperiod and PPF of 60 µmol m -2 s -1 at 25 ± 2 o C. The frequency of 

decontaminated surviving explants, expressed in percentage, in each 

decontamination treatment was recorded. Shoot-tip explants exhibited a high 

decontamination frequency and were therefore selected for all the subsequent 

experiments. Due to the low availability of explants as a result of dormancy 

imposed during the winter season, plant materials were bulked up by subculturing 

on the same medium using screw cap jars (110 mm × 60 mm, approximately 300 

ml volume). Shoot-tip explants obtained from these cultures were used in the next 

two experiments (sections 3.2.2 and 3.2.3) 

3.2.2 Effects of BA and NAA on shoot multiplication 

After bulking up sufficient plant material, an experiment investigating the effects of 

BA with or without NAA supplementation on shoot multiplication was conducted 

using shoot-tip explants (5 mm length). Concentrations of 0.0, 0.5, and 1.0 µM of 

NAA were combined with 1.0, 2.0, 3.0, 4.0 and 5.0 µM BA in a 3 × 5 completely 

randomised factorial design. MS medium without any plant growth regulator was 

included as a control. Three explants were cultured on 30 ml of medium contained 

in each screw cap jar, with a total replicate (observation unit) of twenty-seven 

explants per treatment. Cultures were incubated in a growth room with 16 h light/8 

h dark conditions and 60 µmol m -2 s -1 PPF at 25 ± 2 o C. The total number of 

adventitious shoots produced per cultured explant, number of adventitious shoots 

greater than 10 mm in length, number of adventitious shoots 5 – 10 mm long, and 

percentage of cultured explants producing shoots were recorded after four and six 

weeks of culture. 

3.2.3 Effects of types and concentrations of cytokinins on shoot multiplication 

Concentrations of 1.0, 3.0, 5.0 and 7.0 µM of different aromatic cytokinins (BA, 

Kinetin, mT, mTR, and MemTR) were used in a completely randomised design. 

50

BA and Kinetin were purchased from SIGMA (USA) while mT, mTR, and MemTR 

were obtained from the Laboratory of Growth Regulators, Palacky University and 

Institute of Experimental Botany AS CR, Czech Republic. Three explants were 

cultured on 30 ml medium contained in each screw cap jar, with a total replicate 

(observation unit) of twenty-four explants per treatment. Cultures were incubated 

in a growth room with 16 h photoperiod and 60 µmol m -2 s -1 PPF at 25 ± 2 o C. The 

total number of adventitious shoots produced per cultured explant, number of 

adventitious shoots greater than 10 mm in length, number of adventitious shoots 5 

– 10 mm long, and percentage of cultured explants producing shoots were 

recorded after four and six weeks of culture. In addition, abnormality index, 

calculated as the ratio of abnormal (hyperhydric shoots and shoots with shoot-tip 

necrosis) to normal shoots (BAIRU et al., 2008), was recorded after six weeks of 

culture. 

3.2.4 Effects of photoperiod on shoot multiplication 

Shoot-tip explants used for this experiment were excised from regenerated shoots 

in cultures containing MS medium supplemented with 7 µM MemTR. Based on the 

results obtained from the preceding experiment (section 3.2.3), three shoot-tip 

explants were cultured on 30 ml of MS medium supplemented with 7 µM MemTR 

in screw cap jar. The cultures were placed at 25°C under two different 

photoperiods: continuous light and 16 h light (60 µmol m -2 s -1 PPF in each case). 

Each treatment had a total of twenty-four replicates. The total number of 


greater than 10 mm in length, number of adventitious shoots 5 – 10 mm long, and 

percentage of cultured explants producing shoots were recorded after six weeks of 

culture. 

3.2.5 In vitro rooting of regenerated shoots 

For in vitro rooting of individual regenerated shoots, shoot clusters produced in the 

shoot multiplication stage from each optimal cytokinin concentration were carefully 

separated and cultured on half-strength MS medium with or without 2.5 µM IBA 

supplementation, contained in screw cap jars. The cultures were completely 

51

andomised and maintained in a growth room at 25°C under a 16 h photoperiod 

(60 µmol m -2 s -1 PPF). After four weeks of culture, the number of roots produced 

per shoot, root length and percentage of cultured shoots producing roots were 

recorded. 

3.2.6 Ex vitro rooting and acclimatization 

Regenerated shoots obtained from MS medium supplemented with 7 µM MemTR 

were used in this experiment. Ex vitro rooting of regenerated shoots was 

investigated by pulsing regenerated shoots in different IBA concentrations for five 

hours in the light at 25°C. The IBA concentrations used were 10 -3 , 10 -4 , 10 -5 and 

10 -6 M while distilled water served as the control. After pulsing, the shoots were 

potted in vermiculite and placed in a mist house with about 90% relative humidity 

for three weeks. The acclimatized plants were subsequently transferred to a 

greenhouse and the percentage of surviving plants was recorded in each of the 

treatments after another two weeks. 

For the purpose of acclimatization, agar was carefully washed off the in vitro 

rooted shoots (regenerated on medium with 7 µM MemTR) and the plants then 

potted in a mixture of sand:soil:vermiculite (1:1:1, v/v). The potted plants were 

maintained in the mist house for two weeks before being transferred to a 

greenhouse. The percentage of surviving plants was recorded after an additional 

two weeks. 

3.2.7 Data analyses 

Mean values of the various treatments were subjected, as appropriate, to either 

one way analysis of variance (ANOVA) or student‟s t-test using SPSS version 15.0 

or SigmaPlot 8.0, respectively. The significance level was determined at P = 0.05. 

Where there were significant differences, the means were separated using 

Duncan‟s Multiple Range Test (DMRT). 

52

3.3 Results and discussion 

Figure 3.1 shows the different stages involved in the successful micropropagation 

of Barleria greenii. These include explant selection from the stock plant, culture 

initiation, adventitious shoot regeneration, rooting of regenerated shoots and their 

acclimatization. 

Figure 3.1: In vitro propagation of Barleria greenii. (A) Stock plant. (B) Control 

(MS medium without PGR). (C) Shoot multiplication on MS medium 

supplemented with 3 µM BA. (D) Shoot multiplication on MS 

medium supplemented with 7 µM MemTR. (E) In vitro rooted 

regenerated shoot ready for acclimatization. (F) Three-month-old 

fully acclimatized plant. Bars = 10 mm. 

53

3.3.1 Explant decontamination 

Figure 3.2 shows the effects of different sodium hypochlorite solution treatments 

on decontamination of nodal and shoot-tip explants. The decontamination 

frequency recorded in the nodal explants was generally low, the highest being 

47% decontamination. On the other hand, shoot-tip explants treated with 3.0% 

NaOCl for 30 min and those treated with 3.5% NaOCl for 20 min showed high 

decontamination frequencies of 72 and 74%, respectively. The soaking of the 

shoot-tips in 70% ethanol for 60 sec followed by 3.5% for 20 min proved to be the 

best surface decontamination treatment. 

Frequency of decontamination (%) 

80 

60 

40 

20 

0 

3% NaOCl for 20 min 

3% NaOCl for 30 min 

3.5% NaOCl for 20 min 

Figure 3.2: Effects of sodium hypochlorite (NaOCl) solution treatments on 

explants decontamination. 

Nodal Shoot-tip 

Explant type 

54


The effects of different combinations of BA and NAA concentrations on 

adventitious shoot production after four and six weeks of culture are presented in 

Figures 3.3 and 3.4, respectively. The number of adventitious shoots produced per 

cultured explant, adventitious shoots with a length greater than 10 mm and 

adventitious shoots 5 – 10 mm in length were higher in all the treatments, 

compared to the control. The treatments with BA alone showed higher adventitious 

shoot production when compared to the BA treatments supplemented with NAA 

concentrations. This trend was observed both after four and six weeks of culture. 

In cultures with BA alone, there was an increase in shoot production with 

increased BA concentration, reaching the optimum at 3 µM BA. At supra-optimal 

concentrations, there was a decrease in shoot production. At equimolar supra- 

optimal BA concentrations, an increase in NAA concentration generally gave a 

reduced shoot production. In a similar vein, the frequency of explants producing 

shoots was generally lower in cultures supplemented with NAA compared to 

cultures with BA alone. These results imply that the exogenous application of NAA 

is neither a requirement for adventitious shoot induction nor shoot proliferation 

from shoot-tip explant of this species. In fact, the addition of NAA tends to show an 

antagonistic effect on adventitious shoot production. Auxins are generally known 

to be produced in shoot-tips of plants, from where they are mainly transported 

basipetally. Hence, in this study, the endogenous auxin content in the excised 

shoot-tip explant appears sufficient for shoot induction and growth. A similar 

significant inhibition of NAA on shoot proliferation and growth was reported by 

NUNES et al. (2002) in Cedrela fissilis cultures. According to these authors, the 

addition of NAA to medium containing BA strengthened apical dominance at the 

expense of shoot proliferation. Furthermore, it is possible that the exogenous 

application of NAA in the current study down-regulated cytokinin levels by 

promoting BA metabolic inactivation through N-glucosylation (KAMĺNEK et al., 

1997). The lowering effect of exogenous auxin application on active cytokinin 

levels has also been reported by other researchers (HANSEN et al., 1987; 

BEINSBERGER et al., 1991; ZAŽĺMALOVÁ et al., 1996). 

55

Adventitious shoots per explant 

(n) 

Adventitious shoots > 10 mm length 

(n) 

Adventitious shoots 5 - 10 mm length 

(n) 

Explants producing shoots 

(%) 

5.0 

4.0 

3.0 

2.0 

1.0 

0.0 

2.0 

1.5 

1.0 

0.5 

0.0 

2.0 

1.5 

1.0 

0.5 

0.0 

100 

80 

60 

40 

20 

0 

A 

B 

C 

D 

g 

e 

d 

ab 

ab 

a 

a 

a 

a 

abc 

ab 

a 

abc 

ab 

abcd 

cdef 

bcd 

bcd 

defg 

def 

bcd 

cde 

efg 

cde 

cd 

bcd 

cd 

0.0 0.5 1.0 

NAA concentration (µM) 


greenii after four weeks of culture. Bars with different letters are 

significantly different (P = 0.05) according to DMRT. 

bcde 

bc 

cdef 

cde 

abcd 

abcd 

cdef 

cde 

abcd 

bcd 

bcd 

abc 

defg 

defg 

cde 

cde 

fg 

de 

bcd 

cd 

cd 

Control 

1.0 µM BA 

2.0 µM BA 

3.0 µM BA 

4.0 µM BA 

5.0 µM BA 

56

Adventitious shoots per explant 

(n) 

Adventitious shoots > 10 mm length 

(n) 

Adventitious shoots 5 - 10 mm length 

(n) 

Explants producing shoots 

(%) 

5.0 

4.0 

3.0 

2.0 

1.0 

0.0 

2.0 

1.5 

1.0 

0.5 

0.0 

2.0 

1.5 

1.0 

0.5 

0.0 

100 

80 

60 

40 

20 

0 

A 

B 

C 

D 

e 

c 

d 

ab 

a 

abc 

a 

a 

a 

a 

ab 

a 

abc 

a 

abc 

cd 

b 

bcd 

de d 

b 

b 

0.0 0.5 1.0 



greenii after six weeks of culture. Bars with different letters are 


de 

bc 

cd 

cd 

cd 

d 

d 

bc b 

bcd 

bcd 

cd 

b 

bcd 

bcd 

b 

abcd 

de 

de 

bc 

bc 

de 

bc 

bcd 

bcd 

bcd 

Control 

1.0 µM BA 

2.0 µM BA 

3.0 µM BA 

4.0 µM BA 

5.0 µM BA 

57

3.3.3 Effects of types and concentrations of cytokinins on shoot multiplication 

Table 3.1 shows the effects of different types and concentrations of aromatic 

cytokinins on adventitious shoot production. The lowest and highest adventitious 

shoot production were observed in the control (Figure 3.1B) and the treatment with 

7 µM MemTR (Figure 3.1D) respectively, both after four and six weeks of culture. 

With the exception of BA treatments, an increase in concentration generally 

resulted in an increase in adventitious shoots produced per cultured explant as 

well as in adventitious shoots with a length greater than 10 mm. All the kinetin 

concentrations (including the highest concentration tested) gave a comparatively 

low adventitious shoot production, which was not significantly different from the 

control. This indicates that kinetin is a relatively weak cytokinin (especially when 

compared with BA), perhaps requiring a much higher concentration to achieve a 

significant shoot production in this species. The low effectiveness of kinetin in 

producing a high shoot proliferation compared to BA has also been reported in 

Crossandra infundibuliformis (another Acanthaceae species) and Citrullus lanatus 

by GIRIJA et al. (1999) and COMPTON et al. (1993), respectively. Nevertheless, 

BOGAERT et al. (2006) observed that kinetin (a weak cytokinin) is useful in slowly 

and safely micropropagating some valuable ornamentals such as chimeras, 

without compromising their histogenic stability. 

The plant growth regulator, BA is reported to be among the most effective and 

affordable cytokinins widely used in micropropagation techniques (BAIRU et al., 

2007). The optimum BA concentration for adventitious shoot production was 

reached in the current study at 3 µM (Figure 3.1C; Table 3.1). The abnormality 

index recorded at this concentration was higher than the abnormality index 

recorded in all concentrations of cytokinins (except 5 µM Kinetin, 1 µM BA and 

supra-optimal BA concentrations) used in this study. At supra-optimal BA 

concentrations, adventitious shoot production decreased while the abnormality 

index increased considerably. This response possibly reflects the toxic nature of 

BA in this species. Similar observations with respect to BA toxicity have been 

reported by many researchers for different plant species (KUBALÁKOVÁ and 

STRNAD, 1992; BOGAERT et al., 2006; BAIRU et al., 2007, 2008, 2009). This 

toxicity is often attributed to the more stable nature of BA and its metabolites such 

58

as [9G]BA compared to other cytokinins. DOLEŽAL et al. (2006) however, 

reported that growth inhibition by excessive cytokinin concentrations in tobacco 

callus is at least partly due to the cytokinin inhibition of cyclin-dependent kinase 

activity. Nevertheless, some recent investigations have focussed on evaluating 

other cytokinins (mainly the aromatic ones) with a view to finding a replacement for 

the use of BA in the micropropagation industry. Such replacement would be 

expected, among other things, to essentially improve multiplication frequency 

while maintaining genetic stability of regenerated plants. 

The use of mT and its derivatives has been advocated by some researchers 

(WERBROUCK et al., 1996; BOGAERT et al., 2006; BAIRU et al., 2007) as a 

potential replacement for BA in the micropropagation industry. Some of these 

topolins were evaluated in this study. Unlike in BA treatments, an increase in 

adventitious shoot production per cultured explant and adventitious shoots with 

length greater than 10 mm was generally observed with an increase in the 

concentration of the topolins evaluated (Table 3.1). It is particularly noteworthy that 

the abnormality index recorded in all the topolin treatments (including the higher 

concentrations evaluated) was much lower than the abnormality index recorded at 

the lowest BA concentration. These findings indicate the effectiveness and less 

toxic nature of these topolins at higher equimolar concentrations compared to BA. 

Some factors responsible for the superiority of meta-topolins over BA have been 

discussed by some researchers. As stated by KAMĺNEK et al. (1987b), the 

localised accumulation of mT is prevented by its faster translocation in plant 

tissues. The metabolites of mT and mTR are said to be easily degradable (BAIRU 

et al., 2009). The hydroxyl group in the side chain of meta-topolins makes possible 

the formation of O-glucoside metabolites (WERBROUCK et al., 1996). The O- 

glucosides are considered to be cytokinin storage forms, stable under certain 

conditions but rapidly converted to active cytokinin bases when required 

(PARKER et al., 1978; WERBROUCK et al., 1996). The reversible sequestration 

of the O-glucosides in turn, allows for the continuous availability of cytokinins at a 

physiologically active level over a prolonged period of time, resulting in high shoot 

formation in in vitro cultures (STRNAD, 1997). 

59

Table 3.1: Effects of types and concentrations of cytokinins on adventitious shoot production of Barleria greenii 

Cytokinin 

(µM) 

4 th Week of culture 6 th Week of culture 

Adventitious Adventitious shoots (n) Explants 

shoots/explant 

(n) 

5 – 10 mm 

length > 10 mm length 

Control 0.92 ± 0.06 h 0.42 ± 0.10 cd 0.50 ± 0.10 h 92 

1 BA 2.46 ± 0.24 cdef 0.79 ± 0.17 bcd 1.67 ± 0.27 cdefg 100 

3 BA 3.04 ± 0.50 bc 1.21 ± 0.27 b 1.83 ± 0.38 bcde 96 

5 BA 2.83 ± 0.27 bcd 1.04 ± 0.19 bc 1.79 ± 0.26 bcdef 100 

7 BA 2.13 ± 0.24 cdefg 0.71 ± 0.20 bcd 1.42 ± 0.18 defg 100 

1 Kin 1.00 ± 0.06 h 0.46 ± 0.10 cd 0.54 ± 0.10 h 96 

3 Kin 1.17 ± 0.13 gh 0.58 ± 0.17 bcd 0.58 ± 0.10 h 92 

5 Kin 1.25 ± 0.12 gh 0.38 ± 0.12 d 0.88 ± 0.14 gh 96 

7 Kin 1.58 ± 0.20 fgh 0.63 ± 0.18 bcd 0.96 ± 0.17 fgh 96 

1 mT 1.67 ± 0.21 efgh 0.21 ± 0.10 d 1.46 ± 0.19 defg 100 

3 mT 2.67 ± 0.34 bcde 0.46 ± 0.18 cd 2.21 ± 0.23 bcd 100 

5 mT 2.71 ± 0.30 bcde 0.46 ± 0.19 cd 2.25 ± 0.26 bcd 100 

7 mT 2.83 ± 0.45 bcd 0.75 ± 0.24 bcd 2.08 ± 0.31 bcde 96 

1 mTR 1.21 ± 0.15 gh 0.25 ± 0.09 d 0.96 ± 0.14 fgh 88 

3 mTR 1.90 ± 0.23 defgh 0.29 ± 0.12 d 1.62 ± 0.22 cdefg 100 

5 mTR 3.17 ± 0.45 bc 0.71 ± 0.19 bcd 2.46 ± 0.35 bc 100 

7 mTR 2.46 ± 0.26 cdef 0.46 ± 0.15 cd 2.00 ± 0.26 bcde 100 

1 MemTR 1.42 ± 0.22 fgh 0.17 ± 0.08 d 1.25 ± 0.24 efgh 100 

3 MemTR 3.08 ± 0.61 bc 1.04 ± 0.36 bc 2.08 ± 0.39 bcde 100 

5 MemTR 3.63 ± 0.43 b 1.04 ± 0.20 bc 2.58 ± 0.38 ab 100 

producing 

shoots (%) 

Adventitious Adventitious shoots (n) Explants Abnormality 

shoots/explant (n) 

5 – 10 mm 

length > 10 mm length 

producing 

shoots (%) index (x 10 -1 ) 

0.96 ± 0.07 g 0.33 ± 0.12 de 0.63 ± 0.10 h 92 0.45 

2.79 ± 0.30 bcde 0.92 ± 0.16 bcd 1.88 ± 0.29 cdef 100 1.55 

3.79 ± 0.62 b 1.38 ± 0.25 ab 2.42 ± 0.48 bcd 96 1.52 

3.13 ± 0.28 bc 1.17 ± 0.21 bc 1.96 ± 0.27 cdef 100 4.42 

2.33 ± 0.29 cdef 0.79 ± 0.20 bcde 1.54 ± 0.26 defgh 100 4.36 

1.04 ± 0.07 g 0.33 ± 0.10 de 0.71 ± 0.09 h 96 0.87 

1.17 ± 0.10 fg 0.54 ± 0.13 cde 0.63 ± 0.10h 96 0.38 

1.25 ± 0.09 fg 0.38 ± 0.10 de 0.88 ± 0.13 gh 100 1.54 

1.63 ± 0.18 efg 0.58 ± 0.15 cde 1.04 ± 0.11 fgh 100 0.26 

1.67 ± 0.21 efg 0.21 ± 0.10 e 1.46 ± 0.19 efgh 100 0.86 

2.96 ± 0.44 bcd 0.58 ± 0.21 cde 2.38 ± 0.31 bcd 100 0.29 

3.08 ± 0.36 bcd 0.58 ± 0.20 cde 2.50 ± 0.28 bc 100 0.72 

3.08 ± 0.48 bcd 0.67 ± 0.23 cde 2.42 ± 0.36 bcd 96 0.72 

1.42 ± 0.16 fg 0.33 ± 0.12 de 1.08 ± 0.16 fgh 92 0 

1.90 ± 0.21 defg 0.19 ± 0.09 e 1.71 ± 0.22 defg 100 0.53 

3.71 ± 0.56 b 0.75 ± 0.22 cde 2.96 ± 0.42 b 100 0.23 

2.92 ± 0.30 bcd 0.63 ± 0.13 cde 2.29 ± 0.27 bcd 100 0.29 

1.46 ± 0.26 fg 0.21 ± 0.08 e 1.25 ± 0.28 fgh 100 0.29 

3.38 ± 0.67 bc 1.13 ± 0.31 bc 2.25 ± 0.41 bcd 100 0 

3.88 ± 0.50 b 1.13 ± 0.28 bc 2.75 ± 0.42 bc 100 0.33 

7 MemTR 5.04 ± 0.62 a 1.75 ± 0.32 a 3.29 ± 0.43 a 100 5.88 ± 0.73 a 1.88 ± 0.31 a 4.00 ± 0.53 a 100 0.37 

Means within the same column followed by different letter(s) are significantly different (P = 0.05) according to DMRT. 

60

Furthermore, BOGAERT et al. (2006) reported the histogenic stability and anti- 

senescing activity of MemTR in Petunia hybrida and Rosa hybrida cultures 

respectively. BAIRU (2008) reported increased callus yield with MemTR > mTR > 

mT in the soybean callus bioassay. In the current study, cultures containing 7 µM 

MemTR (Figure 3.1D) gave both the highest adventitious shoot production per 

cultured explant and adventitious shoots with length greater than 10 mm (5.88 ± 

0.73 and 4.00 ± 0.53 shoots per cultured explant, respectively) after six weeks of 

culture. These values were significantly different from every other treatment and 

the control. In addition, the treatment with 7 µM MemTR gave an abnormality 

index of 0.37, a value lower than that observed in the control. In fact, almost all the 

abnormality indices recorded with mTR and MemTR concentrations were lower 

than that of the control, thus suggesting that the observed abnormalities in mTR 

and MemTR treatments could be carry-over effects of BA since the explants were 

originally taken from cultures containing BA. On one hand, the superiority of mTR 

and MemTR might be due to the presence of a ribose at their N 9 position, which 

better protects them against N 9 -glucosylation (WERBROUCK et al., 1996). On the 

other hand, the overall superiority of MemTR might be partly due to the presence 

of the methyl group in its molecular structure. As shown by SCHIMTZ et al. 

(1972), the presence and position of the methyl group could greatly influence the 

activity of a cytokinin. They observed that a shift of the methyl group from the 3- to 

the 2-position in going from dihydrozeatin to dihydroisozeatin resulted in a 70-fold 

decrease in activity, whereas the complete removal of the methyl group as in the 

case of cis-norzeatin resulted in a significant loss of activity (less than a fifth as 

active as cis-zeatin). BAIRU (2008) reported that the addition of a methoxy group 

to ortho-topolin prevents the formation of a hydrogen bond between the OH group 

on the benzyl ring and the nitrogen at the N 1 position, a bond responsible for the 

intermolecular inhibition of cytokinin activity. The author further mentioned that the 

prevention of hydrogen bonding between the N 1 and H 16 atoms by the methyl 

group serves to increase cytokinin activity by maintaining the dihedral angle 

between least-square planes fitted through the purine and phenyl ring. 

61

3.3.4 Effects of photoperiod on shoot multiplication 

Cultures maintained under a 16 h photoperiod gave a higher (though not 

statistically significant) adventitious shoot production than those under continuous 

light (Table 3.2). The production of adventitious shoots with length greater than 10 

mm was significantly (P = 0.01) higher in cultures maintained under a 16 h 

photoperiod than those under continuous light. This implies an improved growth 

rate in cultures placed under a 16 h photoperiod. ECONOMOU and READ (1986) 

similarly observed increased shoot length and quality in azalea (Rhododendron 

sp.) shoot-tip cultures grown under a 16 h photoperiod compared to continuous 

illumination, which inhibited shoot elongation. While the number of shoots 

produced during the first subculture was the same for both 16 and 24 h 

photoperiods, they further observed that continuous light significantly suppressed 

shoot proliferation during the second subculture. The authors gave two likely 

reasons for their observed response. Firstly, the dark periods (following the 16 h 

photoperiod) might promote the synthesis or accumulation of substance(s) which 

stimulate growth during the ensuing 16 h light periods. Secondly, the reduced 

growth in cultures under continuous light could be due to photo-oxidation of 

endogenous IAA resulting from over-exposure to light, which might affect the 

endogenous balance of the growth regulators. It is very likely that the response 

observed in the current study may be due to change in endogenous balance of 

growth regulators as a result of IAA photo-oxidation or synthesis of growth 

promoting substance(s). 

Furthermore, a high accumulation of CO2 in culture vessels/tubes has been 

reported during the dark period of a light: dark cycle (FUJIWARA et al., 1987; 

cited by SERRET et al. 1997). Such a high availability of CO2 could result in an 

increased rate of photosynthesis and reduced photorespiration, at least during the 

early phase of the subsequent light period in cultures maintained under a 16 h 

photoperiod. Photorespiration, a light dependent process, is known to reduce net 

CO2 fixation and subsequent growth rates in C-3 plants (SALISBURY and ROSS, 

1992). The increased CO2 concentration could result in decreased 

photorespiration by increasing the ratio of CO2 to O2 available in a reaction 

involving the enzyme ribulose bisphosphate carboxylase (rubisco), leading to a 

62

faster or an improved net photosynthesis (SALISBURY and ROSS, 1992). In 

addition to the improved growth rate observed in cultures maintained under a 16 h 

photoperiod, the reduced energy consumption can greatly cut down the cost of 

propagating this species in vitro. 

Table 3.2: Effects of photoperiod on adventitious shoot production of Barleria 

Parameter measured 

greenii after six weeks of culture 

Photoperiod 

24 h light 16 h light 

Adventitious shoots per explant (n) 3.71 ± 0.52 5.38 ± 0.81 # 

Adventitious shoots with length > 10 mm (n) 1.81 ± 0.26 3.52 ± 0.53** 

Adventitious shoots 5 – 10 mm in length (n) 1.90 ± 0.32 1.86 ± 0.33 # 

Explants producing shoots (%) 100 100 

Mean values followed by the asterisk ( ** ) indicate a significant difference at P = 

0.01 while # indicates a non-significant difference at P = 0.05 according to t-test. 

3.3.5 In vitro rooting of regenerated shoots 

Figure 3.5 shows the effects of half strength MS medium with or without IBA 

supplementation on the in vitro rooting of regenerated shoots. Generally, the 

number of roots produced per shoot significantly increased with IBA 

supplementation, although there was no significant difference in the root length. 

Similarly, the frequency of shoots producing roots increased in cultures 

supplemented with IBA. There was however, no significant difference both in root 

length and number of roots produced by shoots from different cytokinin treatments. 

The low production of roots in the medium without auxin supplementation might 

possibly be attributed to the low endogenous auxin concentration produced in the 

shoot apex and transported in a basipetal manner to the basal cut surface (DE 

KLERK et al., 1999). On the other hand, the effectiveness of exogenous 

application of IBA in root induction and elongation of many plant species has been 

reported (CHOFFE et al., 2000; FOGAÇA and FETT-NETO, 2005; IAPICHINO 

and AIRÒ, 2008). The auxin IBA can be converted to IAA and is therefore known 

63

to be a slow release reservoir of a more easily metabolized auxin (EPSTEIN and 

LAVEE, 1984). 

Mean no. of roots per shoot 

Mean root length (cm) 

No. of shoots producing roots (%) 

6.0 

5.0 

4.0 

3.0 

2.0 

1.0 

0.0 

2.5 

2.0 

1.5 

1.0 

0.5 

0.0 

80 

60 

40 

20 

0 

A 

B 

C 

bcd 

a 

a 

a 

d 

a 

abc 

a 

BA mT mTR MemTR 

Source of rooted shoot 

cd 

a 

ab 

a 

Figure 3.5: Effects of half strength MS medium with or without IBA treatment on 

in vitro rooting of regenerated shoots. Bars with different letters are 


d 

a 

a 

a 

½ MS 

½ MS + 2.5 µM IBA 

64

3.3.6 Ex vitro rooting and acclimatization 

The effects of pulsing regenerated shoots in different IBA concentrations on ex 

vitro percentage survival is presented in Figure 3.6. It appears that the percentage 

survival increased with increased IBA concentration. The best percentage survival 

(65%) was achieved in the treatment with 10 -3 M IBA. An increase in the pulsing 

period or IBA concentration might likely improve the survival rate recorded in this 

study. Nevertheless, the survival rate recorded in the treatment with 10 -3 M IBA 

was higher than that recorded in the case of acclimatized in vitro rooted shoots 

with 52% survival. In addition, the direct acclimatization of regenerated shoots 

following the pulse treatment with IBA concentration is time-saving and less 

labour-intensive when compared to in vitro rooting followed by acclimatization. It is 

possible that some of the roots produced in vitro are not functional in the normal 

substrate to which they were transplanted, resulting in a cessation of growth and 

subsequent death of some potted plants (DEBERGH and MAENE, 1981). The low 

survival rate recorded during the acclimatization of in vitro rooted shoots might 

also be due to the fragile nature of the roots produced, which are susceptible to 

damage during transplanting and can lead to plant death (DEBERGH and 

MAENE, 1981). In general, however, there was no morphological variation 

observed in any of the acclimatized plants even after 3 months under greenhouse 

conditions (Figure 3.1F). This developed protocol has a potential of producing 

more than 60,000 transplantable shoots per year from a single shoot-tip explant. 

65

Percentage survival 

70 

60 

50 

40 

30 

20 

10 

0 

0 10 -6 

10 -5 

IBA concentration (M) 

Figure 3.6: Effects of pulsing with different IBA concentrations on ex vitro 

acclimatization of regenerated shoots. 

10 -4 

10 -3 

66


Chapter 4 In vitro propagation of Huernia hystrix 

Huernia hystrix (Hook.f.) N.E.Br. (Family: Asclepiadaceae) is a succulent used as 

a medicinal plant, destructively harvested as a whole plant and extensively sold 

and marketed in South Africa. In the recent survey of plants sold by traders at 

Zululand muthi markets, NDAWONDE et al. (2007) listed H. hystrix as one of the 

threatened plant species that are more scarce (below 20%) than others. They 

noted that traders reported a high market demand for this species. OLIVER (1998) 

described Huernia species (including H. hystrix) as interesting container plants for 

ornamental purposes. 

In some cases when the flowers are sterile, this plant does not set seed 

(ANONYMOUS, 2007). In other situations when the flowers are not sterile and 

seeds are eventually produced, the plant does not often come true from seed 

(ANONYMOUS, 2007). The conventional propagation method is therefore by 

cuttings. Since H. hystrix is a dwarf species, very limited cuttings can be taken 

from the mother plant. Vertical planting of the stem cutting disposes the part in the 

rooting substrate to rot rather than to root (HODGKISS, 2004). OLIVER (1998) 

noted that asclepiad species are prone to numerous diseases and are perhaps 

among the most difficult of the succulent group to grow. Owing to these inherent 

propagation problems, HODGKISS (2004) was of the opinion that regular 

propagation with a few cuttings seems to be the best insurance against the loss of 

such asclepiad species. Its conventional propagation cannot meet the increasing 

demand, which will ultimately lead to extinction if no attention is given to its 

conservation and propagation. The need for an effective propagation method that 

can possibly be used as a conservation measure for this species is further 

heightened by it being red-listed and ranked as vulnerable in its global 

conservation status (HILTON-TAYLOR, 1996; SCOTT-SHAW, 1999). 

According to MALDA et al. (1999), nearly all succulent plant species are affected 

by habitat destruction and the collection of wild plants for illicit trade. The 

67

exponential population growth rates in developing countries since the latter half of 

the twentieth century, leading to an increased demand for medicinal plants, has 

further exacerbated this problem (JÄGER and VAN STADEN, 2000). The 

application of micropropagation techniques in the conservation and rapid mass 

propagation of threatened species is well known and has gained tremendous 

impetus in the last two decades. According to SARASAN et al. (2006), many plant 

species belonging to different major taxonomic categories are being tissue 

cultured for propagation and conservation purposes. Nevertheless, the commercial 

application of micropropagation techniques is generally still hampered by high 

production costs. These costs are attributed to high labour costs, low growth rates 

in vitro and poor survival of the plantlets during acclimatization (KOZAI et al., 

1997). Plant growth and development in vitro are affected by a host of factors 

including the environmental and chemical conditions during the culture period. 

Environmental factors such as temperature, photoperiod, light intensity, humidity, 

and gaseous environment affect physiological processes in plants and are 

therefore critical to micropropagation success. 

It is well known that the optimal environmental and chemical conditions for plant 

growth and development often vary between species and sometimes genotypes. 

This therefore necessitates investigating the effects of these factors on in vitro 

growth and development of individual species while developing a 

micropropagation protocol especially amenable to commercial application. Up to 

the point of publishing the results of this study, there is no detailed report, to our 

knowledge, on the micropropagation of any Huernia species. In order to meet the 

increasing demand for H. hystrix while conserving those in the wild, this study was 

aimed at developing a simple, rapid and cost-effective protocol for its clonal 

propagation. The effects of temperature, photoperiod and culture vessel size on 

adventitious shoot production were also investigated in the establishment of the 

protocol. 

68


4.2.1 Source material, decontamination and bulking of explants 

Stock plants planted in pots (Figure 4.1A) were maintained in the shade house in 

the University of KwaZulu-Natal Botanical Garden. Stem explants taken from the 

stock plants were thoroughly washed under running tap water and dipped in 80% 

ethanol for 60 sec prior to additional decontamination treatments. The additional 

decontamination treatments included the use of either 3.5% sodium hypochlorite 

solution, 0.1 or 0.3% (w/v) mercuric chloride solution for 10, 20 or 30 min. A few 

drops of Tween 20 were added to the solution as a surfactant in each case. The 

surface decontaminated stem explants were cut into 10 mm lengths and then 

inoculated on 10 ml (in culture tubes, 100 mm × 25 mm, 40 ml volume) of full 

strength MS medium supplemented with 30 g l -1 sucrose, 0.1 g l -1 myo-inositol, 

2.69 µM NAA, 22.19 µM BA and solidified with 8 g l -1 agar (Bacteriological agar– 

Oxoid Ltd., Basingstoke, Hampshire, England). The pH of the medium was 

adjusted to 5.7 with KOH or HCl before autoclaving at 121°C and 103 kPa for 20 

min. Cultures were incubated in a growth room under continuous light provided by 

cool white fluorescent tubes (Osram ® L 58 W/640, 30 µmol m -2 s -1 PPF) at 25 ± 

1°C. The frequency of surviving decontaminated explants was recorded in 

percentage for each of the decontamination treatments. Based on the results 

obtained from this experiment, stem explants that were surface decontaminated 

using 80% ethanol followed by 0.3% mercuric chloride solution with a few drops of 

Tween 20 for 20 min were cultured on the medium described above for the 

purpose of bulking up more explants. 


Following bulking of sufficient plant material, shoot multiplication experiments were 

designed using stem explants, excluding terminal portions, and cut into 10 mm 

lengths. Concentrations of 0.00, 2.69, 5.37 and 8.06 µM of NAA were combined 

with 4.44, 13.32 and 22.19 µM BA in a 4 × 3 completely randomised factorial 

design. A control without any plant growth regulator was included. Each treatment, 

including the control had 20 replicates and the experiment was repeated twice. 

69

Cultures were incubated under the same growth conditions stated above. After 

nine weeks, the following growth parameters were recorded: the total number of 

adventitious shoots produced per cultured explant (shoot multiplication rate), 

number of adventitious shoots 5 – 10 mm long, number of adventitious shoots 

greater than 10 mm in length, number of adventitious roots per cultured explant, 

fresh weight of the adventitious shoots regenerated from each explant, fresh 

weight of the adventitious roots regenerated from each explant, percentage of 

cultured explants producing shoots and the presence or absence of basal callus. 

4.2.3 Effects of temperature and photoperiod on shoot multiplication 

Stem explants (10 mm) were cultured individually on 10 ml of medium (full 

strength MS medium supplemented with 5.37 µM NAA and 22.19 µM BA) 

contained in culture tubes. The NAA and BA concentrations used were selected 

because they gave the best shoot proliferation in the experiment described in 

section 4.2.2. The cultures were maintained in different growth cabinets under two 

sets of light conditions, defined as follows: (a) 15, 20, 25, 30, and 35°C at 16 h 

photoperiod; (b) 25, 30, and 35°C at 24 h photoperiod. A PPF of 30 µmol m -2 s -1 

provided by cool, white fluorescent tubes (Osram ® L 58 W/640) was used in all the 

experiments. Each treatment had at least 18 replicates. After eight weeks of 

culture, the following growth parameters were recorded: the number of 


5 – 10 mm long, number of adventitious shoots greater than 10 mm in length, 

fresh weight of the adventitious shoots regenerated from each explant and the 

percentage of cultured explants producing shoots. 

4.2.4 Determination of titratable acidity 

On the basis of the results obtained from experiments on effects of temperature 

and photoperiod, nocturnal changes in titratable acidity was determined after eight 

weeks in shoots regenerated from explants cultured under a 16 h photoperiod at 

either 25 or 35 °C. The method described by MARTIN et al. (1990) was followed. 

The objective was to determine the presence of crassulacean acid metabolism 

(CAM) in these cultures. Shoot tissues were collected at the beginning (22:00 h), 

70

middle (02:00 h) and end (06:00 h) of an 8 h dark period, weighed and ground in 

distilled water. The ground tissues were then filtered under vacuum and the filtrate 

was titrated against 0.01 N NaOH to a pH 7.0. Titratable acidity was expressed in 

µM H + g -1 fresh weight. Each determination had four replicates. 

4.2.5 Effects of culture vessel size on shoot multiplication 

Explants were cultured in two culture vessels of different size. Individual stem 

explants were cultured on 10 ml of medium contained in culture tubes (100 mm × 

25 mm, 40 ml volume) while three stem explants were cultured on 30 ml of 

medium contained in tightly-closed screw cap jars (110 mm × 60 mm, 

approximately 300 ml volume). Each culture tube had a headspace volume of 30 

ml per explant while the screw cap jar had a headspace volume of 90 ml per 

explant. The cultures were kept in a growth room maintained at 25°C and 

continuous photoperiod (30 µmol m -2 s -1 PPF). A total of 18 replicates (observation 

unit) per treatment were used. The experiment lasted for eight weeks whereafter 

the same growth parameters described above (section 4.2.3) were recorded. 

4.2.6 Indirect organogenesis 

The callus produced at the base of the stem explant during the bulking process 

was subcultured on the same medium to produce more calli. A completely 

randomised experiment was designed to determine the organogenic potential of 

these calli. Concentrations of 0.00, 0.27, 2.69, and 5.37 µM NAA were combined 

in a factorial manner with 0.00, 2.22, 5.55, and 11.10 µM BA. Cultures were 

incubated in a growth room under continuous light (30 µmol m -2 s -1 PPF) at 25°C. 

Each treatment had 15 replicates. After nine weeks, presence or absence of 

organogenesis was examined and callus fresh weight recorded. 

4.2.7 Rooting and acclimatization 

For rooting of individual shoots, shoot clusters produced in the shoot multiplication 

stage were carefully separated and cultured in PGR free half-strength MS medium 

as well as half-strength MS medium supplemented with 1 µM IBA contained in 

71

loosely closed screw cap jars (300 ml). After four weeks of culture under 

continuous light (30 µmol m -2 s -1 PPF) at 25°C, the rooted shoots were planted in a 

potting mixture of 1:1 (v/v) ratio of soil:sand, treated with fungicide (Benlate, 

0.01%) and transferred to the greenhouse. The greenhouse thermostat was set to 

regulate the temperature at 15°C minimum and 25°C maximum. After two months 

of growth in the greenhouse, fresh and dry weights of shoots and roots, number of 

roots and percentage survival were recorded. 


The data collected were subjected to student‟s t-test or one-way ANOVA where 

appropriate. Where there was a significant difference (P ≤ 0.05), the means were 

further separated using Duncan‟s Multiple Range Test (DMRT). The analysis was 

done using SigmaPlot version 8.0 (t-test) and SPSS software version 15.0 

(ANOVA). 


4.3.1 Explant decontamination 

All the treatments with 3.5% sodium hypochlorite solution were contaminated. The 

frequencies of surviving clean explants obtained in different treatments with 

mercuric chloride solution are presented in Figure 4.2. The treatment with 0.3% 

(w/v) mercuric chloride for 20 min gave the highest frequency of 55% clean 

explants. All the remaining treatments gave a frequency ranging from 0 to 35% 

decontamination. In general, the low frequency observed was due to a high 

incidence of internal contamination of the explants. 

72

Figure 4.1: In vitro propagation of Huernia hystrix. (A) Stock plant. (B) Control 

with root production (MS medium without plant growth regulators). 

(C) Multiple shoot production accompanied with root formation on 

MS medium supplemented with 5.37 µM NAA and 22.19 µM BA. 

(D) Two-month-old fully acclimatized plants (E) Green callus growth 

with root hairs on MS medium supplemented with 5.37 µM NAA. (F) 

Root regeneration from callus on MS medium supplemented with 

2.69 µM NAA and 2.22 µM BA. Scale bar = 10 mm. 

73

Frequency of decontamination (%) 

60 

50 

40 

30 

20 

10 

0 

10 min 

20 min 

30 min 

0.1 0.3 

Mercuric chloride solution (w/v) 

Figure 4.2: Frequencies of explants decontamination in different mercuric 

chloride solution treatments. 

4.3.2 Shoot and root organogenesis 

The effects of different combinations of NAA and BA concentrations on root and 

shoot organogenesis are presented in Table 4.1. All the treatments showed a 

significantly higher adventitious shoot production compared to the control with an 

average of 0.71 ± 0.097 shoots per explant (Figure 4.1B). A maximum of 4.11 ± 

0.428 adventitious shoots per explant with 100% frequency developed on media 

supplemented with 5.37 µM NAA and 22.19 µM BA (Tables 4.1and 4.2, Figure 

4.1C). The same media also produced the highest number of adventitious shoots 

with a length greater than 10 mm. At the same concentration of NAA, adventitious 

shoot production generally increased with increased BA concentrations. Similarly, 

at the same level of BA concentration, adventitious shoot production generally 

increased with increased NAA concentration. It therefore appeared that BA and 

NAA have synergistic effects on shoot multiplication. SUDHA et al. (1998) 

observed that supplementation with NAA in addition to BA was necessary for 

shoot elongation and multiple shoot production from nodal explants of 

Holostemma annulare (an Asclepiadaceae species). 

74

Table 4.1: Effects of different combinations of NAA and BA on shoot and root regeneration of Huernia hystrix after nine weeks of 

culture 

PGR combination 

NAA:BA (µM) 

Adventitious shoots Adventitious roots Adventitious shoots (n) 

per explant (n) per explant (n) 5 - 10 mm length > 10 mm length 

Control 0.71 ± 0.097 f 3.77 ± 0.527 def 0.40 ± 0.093 c 0.31 ± 0.080 f 

0.00:4.44 2.11 ± 0.292 e 1.57 ± 0.313 f 1.17 ± 0.203 ab 0.94 ± 0.192 ef 

0.00:13.32 2.91 ± 0.475 abcde 1.31 ± 0.325 f 1.26 ± 0.270 ab 1.66 ± 0.307 cde 

0.00:22.19 3.46 ± 0.358 abcd 1.17 ± 0.433 f 1.80 ± 0.252 a 1.66 ± 0.333 cde 

2.69:4.44 2.40 ± 0.296 de 8.51 ± 1.319 ab 1.00 ± 0.169 bc 1.40 ± 0.253 de 

2.69:13.32 2.94 ± 0.272 abcde 2.69 ± 0.590 ef 0.89 ± 0.196 bc 2.06 ± 0.275 abcd 

2.69:22.19 3.77 ± 0.480 abc 2.94 ± 1.332 ef 1.89 ± 0.317 a 1.89 ± 0.350 bcd 

5.37:4.44 2.83 ± 0.334 bcde 7.71 ± 1.338 abc 1.34 ± 0.201 ab 1.49 ± 0.230 cde 

5.37:13.32 3.74 ± 0.508 abc 5.89 ± 1.234 bcde 1.46 ± 0.237 ab 2.29 ± 0.375 abcd 

5.37:22.19 4.11 ± 0.428 a 3.91 ± 1.177 def 1.29 ± 0.203 ab 2.83 ± 0.321 a 

8.06:4.44 2.60 ± 0.290 cde 9.89 ± 1.364 a 0.74 ± 0.180 bc 1.86 ± 0.210 bcde 

8.06:13.32 4.06 ± 0.533 ab 6.23 ± 1.184 bcd 1.37 ± 0.266 ab 2.69 ± 0.366 ab 

8.06:22.19 3.57 ± 0.432 abcd 5.03 ± 0.964 cde 1.20 ± 0.187 ab 2.37 ± 0.343 abc 

Mean values followed by different letters in a column are significantly different (P = 0.05) according to DMRT. 

75

Table 4.2: Frequencies of shoot, root and basal callus production from 

treatments with different concentration combinations of NAA and BA 

PGR combination Explants producing Explants producing Frequency of 

NAA:BA (µM) shoots (%) roots (%) basal callus (%) 

Control 80 100 0 

0.00:4.44 95 75 5 

0.00:13.32 100 65 10 

0.00:22.19 100 35 10 

2.69:4.44 100 90 35 

2.69:13.32 100 85 20 

2.69:22.19 100 30 15 

5.37:4.44 95 90 25 

5.37:13.32 100 90 40 

5.37:22.19 100 70 5 

8.06:4.44 100 100 10 

8.06:13.32 100 90 25 

8.06:22.19 100 90 15 

The synergistic effect of BA in combination with an auxin on shoot multiplication 

has been reported for many Asclepiadaceae medicinal plants such as 

Hemidesmus indicus (SREEKUMAR et al., 2000), Holostemma ada-kodien 

(MARTIN, 2002), and Ceropegia candelabrum (BEENA et al., 2003). Increased 

NAA concentration resulted in an increase in the number of adventitious roots 

produced per explant (Table 4.1). However, at the same NAA concentration, an 

increase in BA concentration caused a reduction in both frequency and the 

number of adventitious roots produced per explant (Table 4.2). This could be due 

to a reduction in the NAA:BA ratio since a high auxin:cytokinin ratio is known to 

promote root production. PATNAIK and DEBATA (1996) observed a similar 

response in Hemidesmus indicus when various concentrations of IBA were 

combined with a fixed concentration of kinetin. 

Regenerated shoots remained viable for more than six months when left in the 

original medium without any subculture. This has potential as a cost-effective 

76

short-term in vitro storage of H. hystrix germplasm. Shoot regeneration in all 

treatments with PGR was accompanied by callus formation at the basal cut end of 

the explant (Table 4.2). Various workers have reported similar observations for 

other Asclepiadaceae species (SUDHA et al., 1998; MARTIN, 2002). The prolific 

adventitious shoot production coupled with callus formation at the base of the 

explant may be due to auxin accumulation at the basal cut ends by downward 

movement, which stimulates cell proliferation especially in the presence of 

cytokinins (MARKS and SIMPSON, 1994). 

Table 4.3 shows the effects of different combinations of NAA and BA 

concentrations on fresh weights of regenerated shoots and roots per cultured 

explant. At all NAA concentrations, supplementation with 13.32 µM BA gave the 

highest fresh weight of the shoots regenerated per explant, which was significantly 

different in most cases compared to the control. This was however not significantly 

different from supplementation with 22.19 µM BA. At the same BA concentration, 

fresh weight of the shoots regenerated per explant generally increased with 

increased NAA concentrations. On the other hand, the fresh weight of adventitious 

roots produced per cultured explant was consistently lower with increased 

concentrations of BA at the same level of NAA but increased with increased NAA 

concentrations at the same level of BA. This could be due to the fact that BA acts 

specifically on shoot multiplication while NAA acts directly on cell elongation 

(GABA, 2005). 

4.3.3 Effects of temperature and photoperiod on shoot multiplication 

Tables 4.4 and 4.5 show the effects of temperature and photoperiod on 

adventitious shoot production as well as on shoot fresh weight and percentage of 

explants producing shoots, respectively. Although there was no significant 

difference in the mean number of adventitious shoots produced per explant 

between the different temperature treatments under constant light, the number of 

adventitious shoots 5 – 10 mm long significantly increased with increased 

temperature. Conversely, the number of adventitious shoots greater than 10 mm in 

length as well as the fresh weight of adventitious shoots regenerated per explant 

decreased significantly with increased temperature in constant light. According to 

77

SALISBURY and ROSS (1992), a temperature rise increases the ratio of 

dissolved chloroplastic O2 to CO2 in C-3 species, such that oxygen fixation by 

rubisco (ribulose bisphosphate carboxylase) occurs faster and photorespiration 

indirectly slows growth. The authors further added that at high temperatures, ATP 

and NADPH are not produced fast enough in C-3 plants to allow increases in CO2 

fixation. This might explain the results observed in the cultures incubated under 

constant light. Furthermore, 38% of the regenerated shoots in cultures under 

constant light at 35°C appear bleached. This might be due to solarization, a 

phenomenon described by SALISBURY and ROSS (1992) as a light-dependent 

inhibition of photosynthesis followed by oxygen-dependent bleaching of 

chloroplast pigments. 

Table 4.3: Effects of different concentration combinations of NAA and BA on 

fresh weights of adventitious shoots and roots produced per explant 

PGR combination Fresh weight per explant (mg) 

NAA:BA (µM) Shoot Root 

Control 100 ± 15 d 40 ± 4 ef 

0.00:4.44 130 ± 22 d 40 ± 13 ef 

0.00:13.32 230 ± 84 d 30 ± 23 ef 

0.00:22.19 100 ± 20 d 3 ± 2 f 

2.69:4.44 380 ± 82 bcd 330 ± 53 b 

2.69:13.32 680 ± 148 ab 170 ± 37 cde 

2.69:22.19 410 ± 69 bcd 130 ± 47 def 

5.37:4.44 340 ± 35 cd 360 ± 58 b 

5.37:13.32 680 ± 170 ab 290 ± 60 bc 

5.37:22.19 600 ± 98 abc 200 ± 31 bcd 

8.06:4.44 550 ± 114 abc 640 ± 102 a 

8.06:13.32 820 ± 136 a 350 ± 68 b 

8.06:22.19 550 ± 105 abc 330 ± 59 b 

Mean values followed by different letters in a column are significantly different (P = 

0.05) according to DMRT. 

78

Table 4.4: Effects of temperature and photoperiod on adventitious shoot production of Huernia hystrix after eight weeks of culture 

Temperature 

(°C) 

Adventitious shoots per explant (n) 

Adventitious shoots (n) 

5 - 10 mm length 

> 10 mm length 

16 h light 24 h light 16 h light 24 h light 16 h light 24 h light 

15 0.4 ± 0.20 c ND 

20 1.8 ± 0.31 b ND 

25 2.3 ± 0.40 b 4.2 ± 0.47 a** 

30 2.7 ± 0.48 b 4.1 ± 0.53 a# 

0.2 ± 0.10 c ND 

1.0 ± 0.26 bc ND 

1.5 ± 0.26 b# 1.3 ± 0.25 b 

1.2 ± 0.42 b 1.8 ± 0.24 b# 

0.2 ± 0.13 b ND 

0.8 ± 0.22 ab ND 

0.8 ± 0.25 ab 2.9 ± 0.37 a*** 

1.5 ± 0.49 a 2.3 ± 0.42 a# 

35 4.2 ± 0.74 a 4.2 ± 0.47 a# 2.4 ± 0.41 a 3.4 ± 0.50 a# 1.8 ± 0.58 a# 0.8 ± 0.22 b 

ND = Not determined. Mean values followed by different letters in a column are significantly different (P = 0.05) according to DMRT. 

Mean values in the same row per growth parameter followed by asterisks indicate significance at P = 0.001 (***) or P = 0.01 (**) 

while # = non significant effect according to t-test. 

79

Table 4.5: Effects of temperature and photoperiod on frequency and fresh weight of regenerated shoots per explant of Huernia 

Temperature (°C) 

hystrix after eight weeks of culture 

Shoot fresh weight per explant (mg) Explants producing shoots (%) 

16 h light 24 h light 16 h light 24 h light 

15 ND ND 

20 114 ± 33.6 a ND 

25 192 ± 67.0 a 690 ± 95.6 a*** 

30 249 ± 79.3 a 569 ± 71.6 a** 

28 ND 

83 ND 

89 100 

94 96 

35 192 ± 44.0 a 251 ± 34.0 b# 94 100 

ND = Not determined. Mean values followed by different letters in a column are significantly different (P = 0.05) according to DMRT. 

Mean values in the same row per growth parameter followed by asterisks indicate significance at P = 0.001 (***) or P = 0.01 (**) 

while # = non significant effect according to t-test. 

80

On the other hand, a significant increase in adventitious shoot production with an 

increase in temperature was observed in cultures placed under a 16 h photoperiod 

(Table 4.4). At lower temperatures (15 and 20°C), the shoot proliferation rates 

were low due to slow growth and less differentiation of shoot meristems. Such low 

shoot proliferation at lower temperatures offers an approach for in vitro storage of 

Huernia hystrix germplasm. ISLAM et al. (2005) similarly reported a significant 

effect of temperature on in vitro growth of mint plants (Mentha spp.) with slow 

growth at lower temperature (20°C). Slow growth at lower temperatures is 

economically viable especially at times when labour for transplanting or 

subculturing and greenhouse space are not available (KOZAI et al., 1997). 

However, according to these authors, in vitro storage of plantlets at low 

temperatures for production management should maintain the photosynthetic and 

regrowth abilities of the plantlets while suppressing growth. We observed in this 

study that the proliferation rate and percentage of explants producing shoots 

generally improved when the cultures at low temperature were later incubated at 

25°C under constant light. Furthermore, slow growth at low temperature might 

have a beneficial effect on the genetic stability of the cultures (BARNEJEE and 

DE LANGHE, 1985) since much repeated subculturing on medium containing 

plant growth regulators could give rise to undesirable somaclonal variation. 

In cultures placed under a 16 h photoperiod, the maximum number of adventitious 

shoots produced per explant and percentage of explants producing shoots (4.2 ± 

0.74 and 94% respectively) were observed at 35°C. Percentage of explants 

producing shoots increased with increased temperature. There was no significant 

difference in the fresh weight of adventitious shoots regenerated from each 

explant with increased temperature. Temperature is known to affect 

photosynthesis with varying optimum levels depending on the species type and 

the environmental conditions under which the plant is grown. Often plant cultures 

are maintained in vitro at 25°C which is the optimum temperature for 

photosynthesis in many C-3 plants (SALISBURY and ROSS, 1992). However, 

plants exhibiting CAM are reported to have a higher optimum temperature of 

approximately 35°C (SALISBURY and ROSS, 1992). The increased adventitious 

shoot production at higher temperature (35°C) observed in cultures under a 16 h 

81

photoperiod could therefore be due to the presence of CAM which has been 

reported for Huernia species (WATSON and DALLWITZ, 1992). 

Furthermore, a comparison of cultures grown at 25°C under constant light to those 

under a 16 h photoperiod gives more insight into the physiology of this plant 

species. A significantly higher adventitious shoot production as well as fresh 

weight of the shoots regenerated per explant were observed in cultures placed 

under continuous light compared to 16 h light (Tables 4.4 and 4.5). The 

frequencies of explants producing shoots in cultures placed under 24 and 16 h 

light were 100% and 89% respectively. The increases observed in cultures under 

constant light suggest that Huernia hystrix possesses a facultative C-3 

photosynthetic pathway requiring a longer photoperiod for best vegetative growth. 

On the other hand, the reduction in fresh weight of shoots regenerated per explant 

and shoot production observed in cultures kept under 16 h light might be due to 

the formation of malic acid at night or in the dark. Accumulation of malic acid in the 

dark is characteristic of CAM plants, and is often accompanied by a net loss of 

sugars and starch (SALISBURY and ROSS, 1992). The fact that some succulents 

have the ability to shift between the C-3 and CAM photosynthetic pathways based 

on photoperiod has been reported by some authors (QUIEROZ, 1974; CHENG 

and EDWARDS, 1991). The similar adventitious shoot production observed in 

cultures at 24 h light (25°C) compared to those kept at 35°C (16 h light, Table 4.4) 

suggests that an increased photoperiod could in part compensate for sub-optimal 

temperature. Even then, the higher number of adventitious shoots longer than 10 

mm and higher fresh weight of adventitious shoots regenerated per explant in 

cultures at 24 h light (25°C) compared to any of the cultures at 16 h light make 24 

h photoperiod and 25°C temperature the best environmental conditions for the in 

vitro adventitious shoot production of this species. 

4.3.4 Titratable acidity 

Figure 4.3 presents the titratable acidity at the beginning, middle and end of the 

dark periods in cultures incubated at 25 and 35°C. Generally, the levels of 

titratable acidity at the end of the dark period were significantly higher than at the 

82

eginning. In the case of cultures incubated at 25°C, 35% of the increased 

titratable acidity was recorded by the middle of the 8 h dark period. In contrast, 

cultures incubated at 35°C had 92% of the increased titratable acidity by the 

middle of the dark period. This implies a faster increase in titratable acidity with an 

increase in temperature. Temperature is said to affect CAM performance in many 

ways (LÜTTGE, 2004). For instance, as stated by LIN et al. (2006), the catalytic 

activity of phosphoenolpyruvate carboxylase (PEPC) (an enzyme involved in dark 

fixation of CO2) increases with increasing temperature. BRANDON (1967) 

reported optimum activity of acid-producing enzymes in CAM at 35°C. However, 

FRIEMERT et al. (1988) explained that an increase in temperature increases the 

rate of malic acid efflux from the vacuole, which could lead to the exposure of 

PEPC in the cytosol to increasing malic acid concentrations and its subsequent 

allosteric inhibition. The lack of significant difference of the titratable acidity 

between 25°C and 35°C at the different times of measurement (Figure 4.3) might 

be due to increased sensitivity of PEPC to malate at high night temperature 

(CARTER et al., 1995; LIN et al., 2006). In any case, the nocturnal synthesis and 

accumulation of organic acid which is an essential component of CAM (LÜTTGE, 

2004) further indicates the presence of CAM in cultures kept under a 16 h 

photoperiod. Although a low night temperature and high day temperature is often 

indicated as more favourable to CAM, the expression of CAM under constant 

temperature has been reported (CHENG and EDWARDS, 1991; LÜTTGE and 

BECK, 1992). A combination of a hot day with lower night temperature (not tested 

in this study) might likely enhance the expression of CAM in this species. 

83

Titratable acidity (µM H + g -1 FW) 

400 

300 

200 

100 

0 

22:00 h 

02:00 h 

06:00 h 

b 

ab 

a 

Figure 4.3: Nocturnal titratable acidity in Huernia hystrix shoots cultured at 

different temperatures. 22:00, 02:00 and 06:00 h are the beginning, 

middle and end of 8 h dark period respectively. Bars with different 

letters are significantly different (Ρ = 0.05) according to DMRT. 

4.3.5 Effects of culture vessel size on shoot multiplication 

b 

25 35 

Temperature (°C) 

a 

A higher adventitious shoot production (though not statistically significant) of 5.6 ± 

0.94 shoots per explant was obtained with cultures in screw cap jars (Table 4.6, 

Figure 4.4). In the same vein, the number of adventitious shoots longer than 10 

mm as well as fresh weight of shoots regenerated per explant were higher (not 

significantly) in cultures contained in screw cap jars. ISLAM et al. (2005) were of 

the opinion that larger vessels with larger air volumes create a better gaseous 

composition in the headspace by retarding accumulation of unfavourable gases 

which affect growth and development of plants in cultures. In the current study, the 

headspace volume of 90 ml per explant in the screw cap jars compared to 30 ml 

headspace volume per explant in the culture tubes could have created a more 

favourable gaseous composition in the headspace. However, KOZAI et al. (1997) 

stated that the type of vessel closure could also affect the gaseous composition as 

well as light environment of cultures. Besides the improved adventitious shoot 

production and fresh weight of shoots regenerated per explant, the use of larger 

a 

84

culture vessels in this study is more economical as it conserves space in the 

growth room (Table 4.6). Based on the number of adventitious shoots produced 

per explant in each of the two different vessels (Table 4.6), an additional 679 

shoots per square meter could potentially be produced in the growth room with the 

use of the screw cap jars compared to culture tubes. 

Table 4.6: Effects of culture vessel size on adventitious shoot production of 

Parameters measured 

Huernia hystrix after eight weeks of culture 

Culture vessel 

Culture tube 

(40 ml) 

Screw cap jar 

(300 ml) 

Adventitious shoots per explant (n) 4.4 ± 0.53 5.6 ± 0.94 ns 

Explants producing shoots (%) 100 100 

Adventitious shoots > 10 mm in length (n) 2.8 ± 0.48 3.1 ± 0.63 ns 

Adventitious shoots 5 - 10 mm in length (n) 1.6 ± 0.25 2.5 ± 0.48 ns 

Shoot fresh weight per explant (mg) 560 ± 154 630 ± 168 ns 

Shoots (n) per m 2 space 2750 3429 

ns = Non-significant effect (P = 0.05) according to t-test. 

85

Figure 4.4: Huernia hystrix adventitious shoot production from explants 

4.3.6 Indirect organogenesis 

cultured in different culture vessels. (A) Screw cap jar [300 ml]. (B) 

Culture tube [40 ml]. Scale bar = 10 mm. 

The effects of different combinations of BA and NAA on callus growth are 

presented in Figure 4.5. Generally, callus fresh weight increased (significantly in 

many cases) with increased BA concentration at the same NAA concentration. 

Similarly, there was a significant increase in most cases with increased NAA 

concentration at the same level of BA concentration. This suggests that both NAA 

and BA have synergistic effects on callus growth in H. hystrix. MARTIN (2002) 

reported shoot organogenesis from callus developed at the basal cut ends of the 

node and internode explants of Holostemma ada-kodien on MS medium fortified 

with 1.0 - 2.5 mg l -1 BA. On the other hand, PATNAIK and DEBATA (1996) 

reported no shoot formation from Hemidesmus indicus callus. In this study, there 

was no evidence of shoot induction in all the combinations evaluated. However, 

86

the root induction observed in some cases (Figures 4.1E and F) demonstrates the 

organogenic capacity of the induced callus. There may be a need to test other 

growth regulators for shoot induction from the derived callus and evaluate the 

possibility of inducing somatic embryos. Meanwhile the established callus cultures 

could be a potential alternative source of bioactive secondary metabolite 

production (GIULIETTI and ERTOLA, 1999). 

Mean callus fresh weight (g) 

3.0 

2.5 

2.0 

1.5 

1.0 

0.5 

0.0 

0.00 µM BA 

2.22 µM BA 

5.55 µM BA 

11.10 µM BA 

f f 

0.00 0.27 2.69 5.37 


Figure 4.5: Effects of combinations of NAA and BA concentrations on callus 

growth. Bars with different letters are significantly different (Ρ = 

0.05) according to DMRT. 

4.3.7 Rooting and acclimatization 

f 

f 

f 

e 

d 

de 

d 

c 

b 

b 

The efficiency of half strength MS medium with or without IBA supplementation in 

rooting has been reported for many Asclepiadaceae plants (KOMALAVALLI and 

RAO, 2000; SREEKUMAR et al., 2000; MARTIN, 2002; BEENA et al., 2003). 

PATNAIK and DEBATA (1996) observed that IBA was more effective than IAA for 

the induction of rooting in Hemidesmus indicus. Based on the results from the 

multiplication experiments, regenerated shoots were rooted individually on half 

strength MS medium with or without IBA. Table 4.7 shows the effects of half 

de 

c 

b 

a 

87

strength MS medium with or without supplementation with IBA concentration on 

rooting and acclimatization of regenerated shoots. The PGR-free medium 

produced an average of 7.5 roots per shoot, higher than the IBA treatment, 

although with no significant difference. Similarly, the treatment with IBA did not 

produce a significant difference in both the fresh and dry weights of the roots and 

shoots. Rooted plantlets acclimatized successfully in greenhouse conditions with 

more than 95% survival and no observable morphological abnormalities (Figure 

4.1D). Best root induction and survival of Holostemma annulare (another 

Asclepiadaceae species) on half strength MS medium supplemented with 1.48 µM 

IBA has been reported (SUDHA et al., 1998). KOZAI et al. (1997) stated that the 

relatively high production costs of micropropagation, resulting mainly from high 

labour costs, low growth rate in vitro and poor survival rates of the plantlets during 

acclimatization limits its commercial application. The high number of roots per 

shoot produced on half strength PGR-free medium in H. hystrix with subsequent 

high survival rate (after direct transfer to a greenhouse environment) makes this 

regeneration protocol particularly attractive from an economic point of view as it 

reduces labour and cost of production. 

Table 4.7: Effects of half-strength MS medium with or without IBA 

supplementation on rooting and acclimatization of regenerated plants 

Parameter Medium 

½ MS ½ MS + 1 µM IBA 

Total plantlets potted (n) 60 60 

Survival frequency (%) 96.7 100 

Roots produced per shoot (n) # 7.50 ± 0.764 ns 6.75 ± 0.827 

Root fresh weight per plant (g) # 0.10 ± 0.011 ns 0.10 ± 0.018 

Shoot fresh weight per plant (g) # 2.47 ± 0.270 ns 2.52 ± 0.333 

Root dry weight per plant (mg) # 20 ± 1 ns 20 ± 2 

Shoot dry weight per plant (mg) # 100 ± 10 ns 90 ± 12 

ns = Non-significant effect (P = 0.05) 

# Twelve fully acclimatized 2-month-old plants were used for this analysis. 

88

The successful micropropagation system described here provides an effective 

means for the conservation and rapid clonal propagation, within a short time, of 

endangered H. hystrix. The observations from this study provide insight into the 

physiology of this species when cultured at different temperatures and 

photoperiods. This study highlights the need to investigate the effects of 

environmental conditions when developing efficient micropropagation protocols, 

especially for commercial purposes. Optimizing environmental conditions could 

increase growth rate, reduce labour costs and thus subsequent production costs. 

The increased multiplication rate and cost-effective, easy acclimatization process 

makes this protocol highly advantageous. 

89

Chapter 5 Pharmacological and phytochemical evaluation of 


Barleria species and Huernia hystrix 

The development of new diseases and resistance of many pathogens to currently 

used drugs coupled with the negative side-effects of many of these drugs have 

necessitated the continuous search for potent and efficacious new drugs or drug 

leads with minimal or no side-effects (CRAGG et al., 1997). The plant kingdom 

has been appropriately described as a reservoir of many novel biologically active 

molecules of medicinal value (TALHOUK et al., 2007). The exploration of plant 

resources for their phytochemical and pharmacological potential could lead to 

identifying such novel biologically active compounds. According to CRAGG et al. 

(1997), the continuing threat to biodiversity and the proven record of natural 

products in drug discovery, among others, give compelling reasons for expanding 

the exploration of nature to identify novel active agents as leads for effective drug 

development. 

Southern Africa is known to have a highly diverse and bioresource-rich flora 

estimated to be approximately one-tenth of global plant diversity (ELOFF, 1998a). 

This region is also known to have a high level of endemic plant species (VAN 

STADEN, 2008). The rich floral diversity provides a great bioprospecting 

opportunity for the discovery of potentially new pharmacologically active 

compounds. ELOFF (1998a) reported however, that only a relatively small number 

of plants in this flora have been evaluated for their pharmacological and 

phytochemical potential. Yet, many plants in this region are facing the risk of 

extinction even before they are evaluated for their medicinal properties (SHAI et 

al., 2008). The extinction of such species could lead to the loss of potential 

therapeutic compounds and/or genes capable of being exploited in the 

biosynthesis of new potent pharmaceutical compounds or drugs (RATES, 2001). 

HOSTETTMANN and MARSTON (2002) recommended dual biological and 

chemical screening as the fastest approach in the exploration for new lead 

compounds from plant species. They suggested the use of simple, sensitive and 

90

target-specific pharmacological assays with the capacity to quickly localise the 

chosen activity in plant extracts. Plant extracts often contain different chemicals 

with different pharmacological activities (HOUGHTON et al., 2007). Thus, the use 

of a series of pharmacological tests has been recommended in order to get a 

complete bioactivity spectrum of a plant extract (HOUGHTON et al., 2007). 

Furthermore, since the aetiology of many disease states is often due to more than 

one factor, the use of a series of pharmacological tests might be helpful in 

understanding the mechanism of action involved in the therapeutic effect of a 

particular plant extract (HOUGHTON et al., 2007). 

This study was aimed at exploring the medicinal value of two endangered and 

endemic southern Africa species belonging to different taxonomic categories, B. 

greenii (a shrub) and H. hystrix (a succulent). For comparison purposes, extracts 

from two other Barleria species (B. prionitis and B. albostellata) were included. B. 

albostellata is mainly a horticultural species while B. prionitis is widely used in folk 

medicine to treat infection-related ailments (KOSMULALAGE et al., 2007). The 

leaves of B. prionitis are reportedly used in the treatment of fever, toothache, liver 

ailments, against ulcers and piles as well as in irritation control. The aerial parts 

are used in inflammation treatments and the roots to disperse boils and glandular 

swellings (SINGH et al., 2003; VERMA et al., 2005). Different extracts from 

different parts of these plant species were evaluated in a number of 

pharmacological assays with a view to investigating the possibility of plant-part 

substitution as a conservation strategy against destructive harvesting of these 

species for medicinal purpose. The phytochemical properties of different parts of 

these species were also evaluated. 


5.2.1 Collection of plant materials 

Plant materials were collected during the summer in 2007. Barleria greenii was 

bought from the Indigenous Nursery at Natal Botanical Gardens, Pietermaritzburg, 

B. prionitis from Val Lea Vista Nursery, Pietermaritzburg while B. albostellata and 

Huernia hystrix were collected from the University of KwaZulu-Natal Botanical 

91

Garden, Pietermaritzburg, South Africa. Voucher specimens (S. Amoo 02 NU, S. 

Amoo 04 NU, S. Amoo 03 NU, and S. Amoo 05 NU, respectively) were deposited 

in the University of KwaZulu-Natal Herbarium, Pietermaritzburg. Plant parts were 

dried at 50°C, ground and stored in airtight containers at room temperature in the 

dark. 

5.2.2 Pharmacological evaluation 

5.2.2.1 Preparation of extracts 

Dried, ground plant materials were extracted sequentially with petroleum ether 

(PE), dichloromethane (DCM) and 80% ethanol (EtOH) (20 ml/g) using a 

sonication bath containing ice water for 1 h each. Methanolic (MeOH) extracts 

(used in antioxidant and acetylcholinesterase assays) were obtained by extracting 

plant materials with 50% methanol (20 ml/g) using a sonication bath containing ice 

water for 20 min. In each case, the crude extracts were filtered through Whatman 

No. 1 filter paper and concentrated in vacuo at 40°C using a rotary evaporator. 

The concentrated extracts were air-dried at room temperature. 

5.2.2.2 Antibacterial activity 

Minimum inhibitory concentration (MIC) of extracts for antibacterial activity was 

determined using the micro-dilution bioassay in 96-well microtitre plates as 

described by ELOFF (1998b). Overnight cultures (incubated at 37°C in a water 

bath with orbital shaking) of four bacterial strains: two Gram-positive (Bacillus 

subtilis ATCC 6051 and Staphylococcus aureus ATCC 12600) and two Gram- 

negative (Escherichia coli ATCC 11775 and Klebsiella pneumoniae ATCC 13883) 

bacteria were diluted with sterile Mueller-Hinton (MH) broth (1 ml bacteria/50 ml 

MH). Each crude plant extract was re-dissolved in ethanol to make a concentration 

of 50 mg/ml. One hundred microlitres of each extract were two-fold serially diluted 

with 100 µl sterile distilled water in a 96-well microtitre plate for each of the four 

bacteria. A similar two-fold serial dilution of neomycin (0.1 mg/ml) was used as a 

positive control against each bacterium. One hundred microlitres of each bacterial 

culture were added to each well. The ethanol solvent, distilled water and MH broth 

92

were included as negative controls. The plates were covered with parafilm and 

incubated overnight at 37°C. To indicate bacterial growth, 50 µl of 0.2 mg/ml p- 

iodonitrotetrazolium chloride (INT) were added to each well and the plates were 

further incubated at 37°C for at least 30 min. Bacterial growth in the wells was 

indicated by a change in colour, whereas clear wells indicated inhibition by the 

tested extracts. MIC values were recorded as the lowest concentrations of extracts 

showing clear wells. The minimum inhibitory dilution (MID) (ml/g) indicating the 

volume to which the extract derived from 1 g can be diluted and then still inhibit 

bacterial growth (ELOFF, 2004) was also determined for each extract. The assay 

was repeated twice with two replicates each. 

5.2.2.3 Antifungal activity 

Antifungal activity against Candida albicans (ATCC 10231) was performed using 

the micro-dilution assay (ELOFF, 1998b) modified for fungi (MASOKO et al., 

2007). Four milliliters of sterile saline were added to 400 µl of a 24-h-old Candida 

culture prepared in Yeast Malt (YM) broth. The absorbance was read at 530 nm 

and adjusted with sterile saline to match that of a 0.5 M McFarland standard 

solution. From this stock, a 1:1000 dilution with sterile YM broth was prepared. 

One hundred microlitres of each extract dissolved in 80% ethanol at 50 mg/ml 

were two-fold serially diluted with sterile distilled water in a 96-well microtitre plate. 

One hundred microlitres of the dilute fungal culture were added to each well. 

Amphotericin B was used as a positive control while the 80% ethanol solvent, 

distilled water and YM broth were included as negative controls. The plates were 

covered with parafilm and incubated at 37°C overnight after which 50 µl of INT (0.2 

mg/ml) were added to each well as a growth indicator. The wells remained clear 

where there was inhibition. MIC values were recorded as the lowest 

concentrations that inhibited fungal growth after 48 h. To determine whether the 

activity is fungistatic or fungicidal, 50 µl of YM broth were added to the clear wells 

and further incubated for 24 h after which the minimum fungicidal concentration 

(MFC) was recorded as the last clear well. The MID (ml/g) as well as minimum 

fungicidal dilution (MFD) (ml/g), indicating the volume to which the extract derived 

from 1 g can be diluted and still inhibit the growth of or kill the fungal cells (ELOFF, 

93

2004) respectively, were also determined. The assay was repeated twice with at 

least two replicates each. 

5.2.2.4 Anti-inflammatory activity 

Anti-inflammatory activity was evaluated using the enzyme-based cyclooxygenase 

assays (COX-1 and COX-2) as described by JÄGER et al. (1996) as well as 

ZSCHOCKE and VAN STADEN (2000). Plant extracts were re-dissolved in 

ethanol at a concentration of 10 mg/ml to give a concentration of 0.25 µg/µl in the 

final assay. The cofactor solution was prepared by the addition of 10 ml of 0.1 M 

Tris buffer (pH 8.0) and 100 µl of hematin solution to 3 mg of L-epinephrine (6 mg 

for COX-2) and 3 mg of reduced glutathione. The COX-1 (from ram seminal 

vesicles) or COX-2 (human recombinant) enzyme (Sigma-Aldrich) was, in each 

assay, activated with the co-factor solution and pre-incubated on ice for 5 min. 

Enzyme/co-factor solution (60 µl) was added to 20 µl of plant extract solution (2.5 

µl of plant extract + 17.5 µl distilled water) and pre-incubated for 5 min at room 

temperature. Twenty microlitres of [ 14 C] arachidonic acid (16 Ci/mol, 3 mM) were 

added to this enzyme-extract mixture and incubated at 37°C in a water bath for 10 

min. After incubation, the reaction was terminated by adding 10 µl of 2 N HCl. 

Unlabelled prostaglandins (PGE2:PGF2, 1:1 v/v) (4 µl, 0.2 mg/ml) were then added 

to each sample as a carrier solution. 

The labelled prostaglandins synthesized in the assay were separated from the 

unmetabolized arachidonic acid by column chromatography using silica columns. 

Silica gel (0.063-0.200 mm particle size, Merck) in eluent 1 (hexane:1,4- 

dioxan:acetic acid, 350:150:1 v/v/v) was packed to a height of 3 cm in Pasteur 

pipettes stoppered with glass wool. One millilitre of eluent 1 was added to each of 

the assay mixtures and the mixtures were then applied to the columns. The 

unmetabolized arachidonic acid was eluted with 4 ml of eluent 1 (1 ml at a time) 

and discarded. The labelled prostaglandins were then eluted into scintillation vials 

with 3 ml (1 ml at a time) of eluent 2 (ethyl acetate:methanol, 85:15 v/v). Four 

millilitres of scintillation fluid were added to each vial and the radioactivity was 

counted after 30 min in the dark using a Beckman LS6000LL scintillation counter. 

In each assay, four controls were run. Two were backgrounds in which the 

94

enzyme was inactivated with HCl before adding [ 14 C] arachidonic acid and which 

were kept on ice, and two were solvent blanks. Indomethacin was used as a 

positive control at a concentration of 5 µM for COX-1 and 200 µM for COX-2. The 

percentage inhibition by the extracts was calculated by comparing the amount of 

radioactivity present in the sample to that in the solvent blank, using the following 

equation: 

Inhibition % = 1 − 

DPMsample − DPMbackground 

DPMblank − DPMbackground 

× 100 

where DPM is the disintegrations per minute. Results presented are the mean 

values of two experiments (each experiment in duplicate). 

5.2.2.5 Acetylcholinesterase (AChE) inhibition 

A microtitre plate assay based on the colorimetric method described by ELLMAN 

et al. (1961) and outlined by ELDEEN et al. (2005) was used to determine the 

AChE inhibition activity by the plant extracts. The following buffers were prepared: 

Buffer A (50 mM Tris-HCl, pH 8), Buffer B (50 mM Tris-HCl, pH 8, containing 0.1% 

bovine serum albumin) and Buffer C (50 mM Tris-HCl, pH 8, containing 0.1 M 

NaCl and 0.02 M MgCl2.6H2O). Twenty-five microlitres of each plant methanolic 

extract at a known concentration were two-fold serially diluted with millipore 

distilled water in a 96-well microtitre plate. Twenty-five microlitres of 

acetylthiocholine iodide (ATCI) (15 mM in millipore distilled water), followed by 125 

µl of 3 mM 5,5-dithiobis-2-nitrobenzoic acid (DTNB) (dissolved in Buffer C) and 50 

µl of Buffer B were then added to each microtitre plate well. A similar procedure 

was followed with galanthamine (20 µM) used as a positive control in place of 

plant extract. The absorbance was read every 45 s (three times) at 405 nm using a 

microtitre plate reader (Opsys MR TM , Dynex Technologies). After the readings, 25 

µl of 0.2 U ml -1 AChE enzyme (from electric eels) was added to each well and the 

absorbance was read again every 45 s (five times). The absorbance readings 

before the addition of enzyme were subtracted from the absorbance readings 

taken after the addition of enzyme, to correct any increase in absorbance due to 

spontaneous hydrolysis of the substrate. The rate of reaction was calculated for 

95

each of the plant extracts, the blank and positive control. The percentage inhibition 

by the plant extracts was calculated using the formula: 

Inhibition % = 1 – 

5.2.2.6 Antioxidant activity 

Sample reaction rate 

Blank reaction rate 

5.2.2.6.1 DPPH radical-scavenging activity 

× 100 

Fifteen microlitres of methanolic extracts at different concentrations were diluted 

with methanol to a final volume of 750 µl. The diluted extracts were then added to 

an equal volume of DPPH (1,1-diphenyl-2-picrylhydrazyl) (100 µM in methanol) 

(SHARMA and BHAT, 2009) and the mixtures incubated in the dark at room 

temperature for 30 min. The negative control had methanol in place of the extract 

while ascorbic acid and butylated hydroxytoluene (BHT) were used as positive 

controls. The absorbance was read at 517 nm using a UV–visible 

spectrophotometer (Varian Cary 50, Australia). Blank solutions with methanol in 

place of DPPH were included for each extract, in order to correct any absorbance 

due to extract colour. The spectrophotometer was zeroed with methanol and tests 

were carried out in triplicate. The radical scavenging activity (RSA) was calculated 

using the equation: 

RSA % = 1 − 

extract − blank 

control 

× 100 

where Аextract, Ablank and Acontrol are the absorbances of the extract, blank solution 

and negative control, respectively. The EC50, which is the concentration of the 

extract required to scavenge 50% of DPPH radical, was determined for each 

extract using GraphPad Prism software (version 4.03). The data were log- 

transformed, normalized and fitted into a nonlinear regression for EC50 

determination. 

96

5.2.2.6.2 Ferric reducing power activity 

The reducing power of the extracts was determined according to the method 

described by KUDA et al. (2005) with slight modification. Thirty microliters of each 

extract were two-fold serially diluted with methanol in a 96-well microtitre plate, 

followed by the addition of 40 µl each of 0.2 M potassium phosphate buffer (pH 

7.2) and 1% (w/v) potassium ferricyanide. The mixture was incubated in the dark 

at 50°C for 20 min after which trichloroacetic acid (40 µl, 10% w/v), distilled water 

(150 µl) and FeCl3 (30 µl, 0.1% w/v) were added. The positive controls were 

similarly prepared using ascorbic acid and BHT, while methanol was used in place 

of extract as the negative control. After incubating at room temperature for 30 min, 

the absorbance was read at 630 nm (JONFIA-ESSIEN et al., 2008) using a 

microtitre plate reader. 

5.2.2.6.3 β-Carotene linoleic acid assay 

The method described by AMAROWICZ et al. (2004) was followed with slight 

modification. β-Carotene (10 mg) was initially dissolved in chloroform (10 ml) and 

the chloroform was removed by evaporation under vacuum, followed by the 

addition of linoleic acid (200 µl) and Tween 20 (2 ml). The mixture was then made 

up to 500 ml (with aerated distilled water) with vigorous shaking to form an 

emulsion. An aliquot (4.8 ml) of the emulsion was added to 200 µl of plant extract 

(6.25 mg/ml) or BHT (6.25 mg/ml), used as a positive control. Each sample was 

prepared in triplicate and the test systems were incubated in a water bath at 50°C 

for 120 min. Initial absorbance (before incubation) and absorbance at every 30 min 

(during incubation) were read using a UV-visible spectrophotometer (Varian Cary 

50, Australia) set at 470 nm. A Tween 20 solution was used to blank the 

spectrophotometer (PAREJO et al., 2002). Antioxidant activities were expressed 

as ANT (%), ORR and AA (%) at t = 60 or 120 min (AMAROWICZ et al., 2004; 

PAREJO et al., 2002) using the following equations: 

ANT % =

ORR =

sample was tested in three replicates and the results were expressed in mg or µg 

gallic acid equivalents (GAE) per gram dry weight (DW). 

5.2.3.3 Total iridoid content 

The total iridoid content was determined following the method described by 

LEVIEILLE and WILSON (2002), which was adapted from HAAG-BERRURIER et 

al. (1978). The method was based on the characteristics of glucoiridoids to form a 

fulvoiridoid complex when reacted with aldehydes (such as vanillin) in an acidic 

medium (LEVIEILLE and WILSON, 2002). A vanillin-sulphuric acid reagent was 

prepared by the addition of methanol (82 ml), vanillin (100 mg) and concentrated 

sulphuric acid (8 ml). A blank reagent containing methanol (82ml) and 

concentrated sulphuric acid (8 ml) was also prepared. In triplicate, the vanillin- 

sulphuric acid reagent (1.35 ml) was added to 150 µl of each extract. A blank was 

prepared for each extract by adding 1.35 ml of the blank reagent to 150 µl of 

extract. The reaction occurred immediately at room temperature and the 

absorbance was read at 538 nm using a UV–visible spectrophotometer (Varian 

Cary 50, Australia). HPLC-grade harpagoside (Extrasynthèse, France) was used 

as a standard for the calibration curve. The total iridoid content, expressed in µg 

harpagoside equivalents (HE) per gram DW was determined for each extract using 

their differential absorbance values correlated with the calibration curve. 

5.2.3.4 Flavonoid content 

The vanillin assay as described by NDHLALA et al. (2007) was used to determine 

the flavonoid content of the samples. Fifty microlitres of each extract were diluted 

with distilled water to 1 ml before the addition of 2.5 ml of methanol-HCl (95:5 v/v) 

and finally 2.5 ml of vanillin reagent (1% w/v). The mixtures were incubated for 20 

min at room temperature after which the absorbance was read at 500 nm using a 

UV–visible spectrophotometer (Varian Cary 50, Australia) against a blank 

containing methanol instead of plant extracts. Each extract had three replicates. 

Standards for the calibration curve were prepared using catechin and the flavonoid 

content was expressed in mg catechin equivalents per gram DW. 

99

5.2.3.5 Gallotannin content 

The rhodanine assay method as described by MAKKAR (2000) was used to 

determine the gallotannin content. Fifty microlitres of each plant sample were 

diluted with distilled water to 1 ml. One hundred microlitres of 0.4 N sulphuric acid 

and 600 µl of rhodanine solution (0.667% w/v in methanol) were added to the 

diluted extract. Two hundred microlitres of 0.5 N KOH were then added after 5 min 

of incubation at room temperature, and 4 ml distilled water was added after a 

further 2.5 min. The mixtures were further incubated at room temperature for 

another 15 min after which the absorbance was read at 520 nm using a UV–visible 

spectrophotometer (Varian Cary 50, Australia) against a blank prepared similarly 

but containing methanol instead of plant extracts. Each extract had three 

replicates. Gallic acid was used to prepare the standards for the calibration curve 

and gallotannin content was expressed in µg gallic acid equivalents (GAE) per 

gram DW. 

5.2.3.6 Condensed tannin (proanthocyanidin) content 

This was determined using the butanol-HCl method as described by MAKKAR 

(2000). Three millilitres of butanol-HCl (95:5 v/v) were added to 500 µl of each 

sample, followed by 100 µl of ferric reagent (2% w/v ferric ammonium sulphate in 2 

N HCl). The mixtures were placed in a boiling water bath for 60 min. The 

absorbance was then read at 550 nm using a UV–visible spectrophotometer 

(Varian Cary 50, Australia) against a blank prepared in a similar way but without 

heating. Each sample had three replicates. Condensed tannins (% per dry matter) 

as leucocyanidin equivalents were calculated using the formula described by 

PORTER et al. (1986): 

Condensed tannins % per dry matter = 

where A550nm is the absorbance value at 550 nm. 

A550nm × 78.26 × Dilution factor 

% dry matter 

100


The percentage inhibitions were log-transformed before they were subjected to 

statistical analysis. Data were subjected to one-way ANOVA. Where there were 

significant differences (P = 0.05), the mean values were further separated using 

DMRT. The analysis was done using SPSS software (version 15.0). 


5.3.1 Yield of plant extracts 

Tables 5.1 and 5.2 show the percentage yield obtained from different extracts of 

Barleria species and Huernia hystrix, respectively. Both the EtOH and MeOH plant 

extracts (polar extracts) have higher yields compared to the PE and DCM extracts 

(non-polar extracts). The yields obtained from the EtOH and MeOH extracts of 

Barleria species leaves were higher compared to other parts of the same plant. In 

H. hystrix, the yield from MeOH extracts were greater than that obtained with 

EtOH extract. 

5.3.2 Pharmacological evaluation 

5.3.2.1 Antibacterial activity 

The MIC and MID of extracts from different parts of the studied Barleria species 

are presented in Table 5.3. All the extracts showed a broad-spectrum antibacterial 

activity. The DCM extract of B. greenii roots gave the best antibacterial activity 

against B. subtilis and S. aureus with MIC values of 59 µg/ml and 234 µg/ml 

respectively. The PE, DCM and EtOH root extracts of B. greenii had lower MIC 

values (in most cases) against all the bacterial strains compared to its stem 

extracts. However, the harvesting of the roots of this endangered plant is 

destructive and could spell more problems for the survival of this species if no 

effective propagation measure is put in place. The observed antibacterial activity 

(especially in the roots) highlights the need for the propagation of this species in 

order to fully explore its potential therapeutic antibacterial agents. In all the 

101

Barleria species, the EtOH extracts had the highest MID values. The DCM and 

EtOH extracts of B. prionitis leaves had lower MIC values against all the bacterial 

strains compared to similar extracts from its stems. This observation suggests the 

potential of substituting the leaves of this plant for its stems. The observed activity 

in B. prionitis is in agreement with the findings of KOSMULALAGE et al. (2007) 

who reported antibacterial activity of ethanolic extracts of aerial parts of B. prionitis 

against S. aureus and Pseudomonas aeruginosa. They reported the isolation of a 

new compound, balarenone along with other known natural products, pipataline 

and 13,14-seco-stigmasta-5,14-diene-3-β-ol from the ethanolic extract, all of which 

showed antibacterial activity against Bacillus cereus and P. aeruginosa. The 

observed antibacterial activity in B. greenii and B. albostellata could be due to the 

presence of these or other compounds. According to JÄGER et al. (1996), when 

active compounds are found in one species, it is likely that more species of the 

same genus contain active compounds of a similar nature. 

Table 5.4 presents the MIC and MID recorded in different extracts of H. hystrix. 

Although a broad spectrum activity with MIC values ranging from 0.39 to 6.25 

mg/ml was observed in all the extracts, only the root extracts showed good activity 

(< 1 mg/ml) against the two Gram-positive bacteria used. The PE extract of the 

roots had lower MIC values against the two Gram-positive bacteria compared to 

the stem and whole plant extracts, indicating its better antibacterial activity. The 

root PE extract also had high MID values against the two Gram-positive bacteria. 

In general, with some exception in root extracts, the MID recorded in ethanol 

extracts was generally higher than all the other extracts. 

102

Table 5.1: Yield (% w/w) of extracts prepared from different parts of three 

Barleria species in terms of starting crude material 

Plant species Plant part Extract Yield (% w/w) 

B. prionitis Leaves PE 0.706 

DCM 0.312 

EtOH 9.404 

MeOH 31.82 

Stems PE 0.362 

DCM 1.578 

EtOH 5.592 

MeOH 9.84 

Roots PE 0.224 

DCM 0.332 

EtOH 4.28 

MeOH 12.05 

B. greenii Leaves PE 2.163 

DCM 0.773 

EtOH 20.565 

MeOH 29.07 


DCM 0.758 

EtOH 8.504 

MeOH 4.53 


DCM 0.492 

EtOH 13.293 

MeOH 12.35 

B. albostellata Leaves PE 5.54 

DCM 1.866 

EtOH 23.306 

MeOH 24.38 


DCM 0.35 

EtOH 5.306 

MeOH 4.56 

103

Table 5.2: Yield (% w/w) of extracts prepared from different parts of Huernia 

hystrix in terms of starting crude material 

Plant part Extract Yield (% w/w) 


DCM 1.896 

EtOH 18.655 

MeOH 34.49 


DCM 1.298 

EtOH 5.428 

MeOH 15.83 

Whole plants PE 1.66 

DCM 1.895 

EtOH 19.577 

MeOH 33.72 

The comparatively lower activity against Gram-negative bacteria by some extracts 

might be as a result of the thick murein layer in these bacteria structures 

preventing the entry of inhibitors (MATU and VAN STADEN, 2003). The low 

potency values generally observed in some of the extracts could be due to the fact 

that the extracts are still in an impure form and thus there could be some 

compounds having an antagonistic effect on the active principle(s). In addition, it 

could be as a result of low concentrations of active compounds present in the 

extracts (RABE and VAN STADEN, 1997). The weak activities observed in vitro 

with some extracts however does not necessarily translate to a similar low activity 

in vivo. Some of these plant extracts, as with some drugs, may be more potent in 

vivo due to metabolic transformation of their components into highly active 

intermediates or their interaction with the immune system (GARCIA et al., 2003; 

NGEMENYA et al., 2006). 

104

Table 5.3: Antibacterial activity (MIC and MID) of crude extracts from different parts of three Barleria species 

Plant species Plant 

part 

Extract Minimum inhibitory concentration (MIC) 

(mg/ml) 

B. s # S. a E. c K. p 

B. prionitis Leaves PE 3.125 3.125 3.125 3.125 

DCM 0.781* 1.563 1.563 1.563 

EtOH 3.125 3.125 3.125 3.125 

Stems PE 1.563 3.125 1.563 3.125 

DCM 3.125 6.250 3.125 3.125 

EtOH 6.250 6.250 6.250 6.250 

B. greenii Stems PE 3.125 6.250 3.125 3.125 

DCM 1.563 1.563 3.125 3.125 

EtOH 6.250 6.250 6.250 3.125 

Roots PE 0.780 3.125 3.125 1.563 

DCM 0.059 0.234 1.875 3.750 

EtOH 0.390 0.781 6.250 3.125 

B. albostellata Leaves PE 0.781 3.125 3.125 1.563 

DCM 0.195 1.563 3.125 0.781 

EtOH 1.563 1.563 3.125 3.125 

Stems EtOH 1.563 3.125 3.125 3.125 

Neomycin (µg/ml) 0.098 1.563 3.125 1.563 

Minimum inhibitory dilution (MID) (ml/g) 

# B. s = Bacillus subtilis, S. a = Staphylococcus aureus, E. c = Escherichia coli, K. p = Klebsiella pneumonia. 

*Values boldly-written are considered very active (< 1 mg/ml). 

B. s S. a E. c K. p 

2.259 2.259 2.259 2.259 

3.995 1.996 1.996 1.996 

30.093 30.093 30.093 30.093 

2.316 1.158 2.316 1.158 

5.050 2.525 5.050 5.050 

8.947 8.947 8.947 8.947 

5.619 2.810 5.619 5.619 

4.850 4.850 2.426 2.426 

13.606 13.606 13.606 27.213 

4.000 0.998 0.998 1.996 

83.390 21.026 2.624 1.312 

340.846 170.205 21.269 42.538 

70.935 17.728 17.728 35.445 

95.692 11.939 5.971 23.892 

149.111 149.111 74.579 74.579 

33.948 16.979 16.979 16.979 

105

Table 5.4: Antibacterial activity (MIC and MBC) of crude extracts from different parts of Huernia hystrix 

Plant part Extract 

Minimum inhibitory concentration (MIC) 

(mg/ml) 

Minimum inhibitory dilution (MID) (ml/g) 

B. s # S. a E. c K. p B. s S. a E. c K. p 

Stems PE 1.56 3.13 3.13 3.13 

DCM 3.13 1.56 3.13 3.13 

EtOH 6.25 6.25 3.13 3.13 

Roots PE 0.78* 0.39 6.25 3.13 

DCM 0.78 1.56 6.25 6.25 

EtOH 6.25 6.25 6.25 3.13 

Whole plants PE 3.13 3.13 6.25 6.25 

DCM 3.13 1.56 3.13 3.13 

EtOH 6.25 6.25 3.13 3.13 

Neomycin (µg/ml) 0.195 0.78 3.13 1.56 

8.256 4.115 4.115 4.115 

6.058 12.154 6.058 6.058 

29.848 29.848 59.601 59.601 

34.974 69.949 4.365 8.716 

16.641 8.321 2.077 2.077 

8.685 8.685 8.685 17.342 

5.304 5.304 2.656 2.656 

6.054 12.147 6.054 6.054 

31.323 31.323 62.546 62.546 

# B. s = Bacillus subtilis, S. a = Staphylococcus aureus, E. c = Escherichia coli, K. p = Klebsiella pneumonia. 


106

5.3.2.2 Antifungal activity 

Opportunistic fungal infections such as candidiasis caused by C. albicans, have 

been reported to be common especially among immunocompromised persons with 

AIDS (acquired immune deficiency syndrome) all over the world (MOTSEI et al., 

2003; SHAI et al., 2008). Table 5.5 shows the antifungal activity of extracts from 

different parts of Barleria species against C. albicans. In general, all the extracts 

demonstrated fungistatic activity against C. albicans. The MIC of the extracts 

ranged from 0.78 to 9.375 mg/ml while the MFC ranged from 1.17 to 12.5 mg/ml. 

The EtOH extracts had the highest MID and MFD in all cases. The PE, DCM and 

EtOH extracts of B. greenii leaves had higher inhibitory activity compared to 

similar extracts of the stems. In the same vein, the leaf EtOH extract of B. greenii 

had the highest MID compared to all other extracts and demonstrated a fungicidal 

activity similar to the EtOH extracts of its roots and stems. The fungistatic activity 

of B. greenii leaf EtOH extract was also higher than the EtOH extract of its stems 

or roots. These results suggest the potential of B. greenii leaves in plant part 

substitution. The leaves can be sustainably harvested for medicinal purposes 

without the destructive harvesting of the roots which could threaten the survival of 

this already rare plant species (ZSCHOCKE et al., 2000b; MATU and VAN 

STADEN, 2003). 

The antifungal activity of H. hystrix extracts against C. albicans is presented in 

Table 5.6. All the extracts showed inhibitory activity with MIC ranging from 0.78 to 

6.25 mg/ml. With the exception of stem EtOH extract (the MFC of which was not 

observed within the concentration range tested), all the extracts evaluated 

generally demonstrated a moderate fungicidal activity against C. albicans. The 

EtOH extracts had the highest MID compared to PE and DCM extracts. The EtOH 

extracts similarly had the highest MFD (except stem EtOH extract) compared to 

PE and DCM extracts. Although the PE and DCM extracts of the whole plant had 

the same MFC as the stem extracts, the inhibitory activities of the whole plant PE 

and DCM extracts were higher than that of the stem or the root. This observation 

possibly suggests the presence of fungistatic agents, likely acting in an additive 

manner in the whole plant extract compared to their individual inhibitory action in 

the stem or root extract. 

107

Table 5.5: Antifungal activity of crude extracts from different parts of three Barleria species against Candida albicans 

Plant species 

Plant 

part Extract 

Minimum inhibitory Minimum fungicidal Minimum inhibitory Minimum fungicidal 

concentration (MIC) (mg/ml) concentration (MFC) (mg/ml) dilution (MID) (ml/g) dilution (MFD) (ml/g) 

B. prionitis Stems PE 3.125 4.688 1.158 0.772 

DCM 3.125 4.688 5.050 3.366 

EtOH 6.250 6.250 8.947 8.947 

Roots PE 4.688 4.688 0.478 0.478 

DCM 2.343 4.688 1.417 0.708 

EtOH 6.250 6.250 6.848 6.848 

B. greenii Leaves PE 7.813 12.500 2.768 1.730 

DCM 0.975* 9.375 7.928 0.825 

EtOH 3.515 9.375 58.506 21.936 

Stems PE 9.375 12.500 1.873 1.405 

DCM 6.250 12.500 1.213 0.606 

EtOH 6.250 9.375 13.606 9.071 

Roots PE 3.125 4.688 0.998 0.666 

DCM 3.125 3.125 1.574 1.574 

EtOH 6.250 9.375 21.269 14.179 

B. albostellata Leaves PE 4.688 6.250 11.817 8.864 

DCM 1.170 4.688 15.949 3.980 

EtOH 4.688 6.250 49.714 37.29 

Stems PE 0.780 1.560 5.103 2.551 

DCM 0.780 1.170 4.487 2.991 

EtOH 3.125 3.125 16.979 16.979 

Amphotericin B (µg/ml) 0.048 0.193 


108

Table 5.6: Antifungal activity of different parts of Huernia hystrix against Candida albicans 

Plant part Extract 

Minimum inhibitory 

concentration (MIC) 

(mg/ml) 

Minimum fungicidal 

concentration (MFC) 

(mg/ml) 

Minimum inhibitory 

dilution (MID) (ml/g) 

Stems PE 1.56 3.125 8.256 4.122 

DCM 1.56 6.25 12.154 3.034 

EtOH 6.25 > 12.5 29.848 ND 

Roots PE 6.25 9.375 4.365 2.91 

DCM 6.25 9.375 2.077 1.385 

EtOH 4.688 4.688 11.578 11.578 

Whole plants PE 0.585 3.125 28.376 5.312 

DCM 0.78* 6.25 24.295 3.032 

EtOH 6.25 6.25 31.323 31.323 

Amphotericin B (µg/ml) 0.048 0.193 

ND = Not determined 

*Value boldly-written is considered very active (< 1 mg/ml). 

Minimum fungicidal 

dilution (MFD) (ml/g) 

109

5.3.2.3 Anti-inflammatory activity 

Figure 5.1 presents the anti-inflammatory activity of different extracts of Barleria 

species as measured by their ability to inhibit COX enzymes. Twelve out of twenty- 

one crude extracts evaluated showed good activity (> 70%) in the COX-1 assay 

while ten extracts showed good activity in the COX-2 assay. All the PE extracts 

(except B. prionitis stem) showed good activity in the COX-1 assay. It is 

noteworthy that all B. greenii extracts showed a consistently higher anti- 

inflammatory activity, significant (P = 0.05) in some cases, compared to B. prionitis 

which is reportedly used in traditional medicine. Both B. greenii and B. albostellata 

have no recorded usage in traditional medicine. In addition, only root EtOH 

extracts of B. greenii out of all the EtOH extracts gave a very good (> 80%) anti- 

inflammatory activity in both COX-1 and COX-2 assays. In general, however, the 

non-polar extracts (PE and DCM) showed better activity compared to the EtOH 

extracts. 

Figure 5.2 shows the anti-inflammatory activity of extracts from different parts of H. 

hystrix. All the PE (except root PE) and DCM extracts consistently showed a high 

activity (> 70%) against both COX-1 and COX-2 enzymes. The EtOH extracts 

generally showed a moderate (40-69%) to poor activity (< 40%) against both 

enzymes. 

COS et al. (2006) observed that compounds that are potent inhibitors of enzymes 

in vitro frequently fail to demonstrate similar activity in vivo because, among other 

things, they do not pass through the cell membrane. The activity shown by the 

non-polar extracts in the current study is therefore of interest since lipophilic 

compounds extractable by non-polar solvents have better resorption through the 

cell membrane (ZSCHOCKE and VAN STADEN, 2000). The inhibition of COX 

enzyme is known to up-regulate the LOX pathway, resulting in the production of 

leukotrienes, which are suggested to be responsible for many of the undesirable 

adverse effects associated with the use of NSAIDs (FIORUCCI et al., 2001; LI et 

al., 2006). Dual inhibitors of COX and LOX enzymes have been suggested to be 

potential classical anti-inflammatory agents with reduced adverse effects 

(ZSCHOCKE et al., 2000a; VIJI and HELEN, 2008). Further evaluation of the 

110

inhibitory activity of the extracts tested in this study against LOX enzyme may 

therefore be needed. 

Percentage prostaglandin synthesis inhibition (COX-1) 

120 

100 

80 

60 

40 

20 

0 

100 

80 

60 

40 

20 

0 

100 

80 

60 

40 

20 

0 

A Leaves Stems Roots 

a a a 

a 

C 

E 

bc 

cd 

bc 

cd 

f 

a 

b 

c 

cd 

cd 

d d 

B.p B.a B.p B.g B.a B.p B.g 

c 

Plant species 

b 

de 

ab 

a 

Percentage prostaglandin synthesis inhibition (COX-2) 

120 B Leaves Stems Roots 

Figure 5.1: Anti-inflammatory activity of extracts from different parts of three Barleria 

100 

100 

species in COX-1 (on the left side) and COX-2 (on the right side) assays. B.p, 

B.a, and B.g are Barleria prionitis, B. albostellata and B. greenii, respectively. 

(A) and (B) are PE extracts. (C) and (D) are DCM extracts. (E) and (F) are 

EtOH extracts. Bars in the same graph bearing different letters are 

significantly different (P = 0.05) according to DMRT. Percentage inhibition by 

indomethacin in COX-1 was 63.4 ± 1.98 and COX-2 was 73.6 ± 1.47. 

80 

60 

40 

20 

0 

80 

60 

40 

20 

0 

100 

80 

60 

40 

20 

0 

D 

F 

e 

c 

bc 

c 

d 

e 

a 

b 

b 

a 

d 

Plant species 

c 

c 

ab 

c 

d d 

cd cd 

B.p B.a B.p B.g B.a B.p B.g 

a 

a 

111

Prostaglandin synthesis inhibition (%) 

100 

80 

60 

40 

20 

0 

80 

60 

40 

20 

0 

A 

B 

a 

a 

Plant extract 

Figure 5.2: Anti-inflammatory activity of different extracts of Huernia hystrix. (A) 

COX-1 assay. (B) COX-2 assay. Bars with different letters in each 

graph are significantly different (P = 0.05) according to DMRT. 

Percentage inhibition by indomethacin in COX-1 was 54.73 ± 2.00 

and COX-2 was 63.44 ± 2.52. 

5.3.2.4 Acetylcholinesterase inhibition 

b 

a 

a 

a 

ab 

a 

b 

PE DCM EtOH 

a 

ab 

c c 

Stems 

Roots 

Whole plants 

Figure 5.3 shows the AChE inhibition activity observed in the MeOH extracts of 

Barleria species. All the extracts evaluated showed a dose-dependent inhibition. In 

general, at the highest extract concentration (0.625 mg/ml), the leaf and stem 

extracts of both B. greenii and B. prionitis exhibited higher inhibitory activities than 

c 

cd 

b 

c 

d 

112

their root extracts. The leaf extracts of B. greenii and B. albostellata showed the 

highest (68%) and lowest (22%) AChE inhibition respectively, at the highest 

extract concentration evaluated. In the same vein, at the lowest extract 

concentration (0.156 mg/ml), the leaf extracts of B. greenii and B. albostellata 

showed the highest (38%) and lowest inhibition (3%) respectively, compared to 

other extracts. The inhibitory activity shown by the leaf extract of B. greenii at the 

lowest extract concentration was in fact significantly higher (P = 0.05) than the 

activities recorded in the stem and root extracts of the same species at the lowest 

concentration. The findings indicate that B. greenii leaves can potentially be 

substituted for its stems or roots in the inhibition of the AChE enzyme. In B. 

albostellata, the inhibitory activity demonstrated by the stem at the lowest extract 

concentration was significantly greater than the activity recorded in its leaf extract. 

This observation suggests that the idea of plant part substitution may be species 

and/or biological activity dependent. 

Acetylcholinesterase inhibition (%) 

80 

60 

40 

20 

0 

efghi 

cdef 

Leaves Stems Roots 

cd 

cdefg 

cde 

a 

j 

hi 

fghi 

B. p B. g B. a B. p B. g B. a B. p B. g 

Plant species 

Figure 5.3: Dose-dependent acetylcholinesterase inhibition by different parts of three 

Barleria species. B.p = Barleria prionitis, B.g = Barleria greenii, B.a = 

Barleria albostellata. Bars bearing different letters are significantly 

different (P = 0.05) according to DMRT. The AChE inhibition activities by 

galanthamine at 0.5, 1.0 and 2 µM were 49.24, 59.81 and 77.03%, 

respectively. 

cde 

hij 

bc 

cdefg 

ij 

ab 

ghi 

cdefg 

a 

efghi 

defgh 

cde 

j 

fghi 

cde 

0.15625 mg/ml 

0.3125 mg/ml 

0.625 mg/ml 

113

The AChE inhibitory activity of extracts from different parts of H. hystrix is 

presented in Figure 5.4. At all the extract concentrations evaluated, the whole 

plant extract showed a higher inhibitory activity, statistically significant in some 

cases, compared to the extracts from the stems or roots. It is likely that the whole 

plant extract contains some compounds that act synergistically or additively to 

produce a higher inhibitory activity compared to their acting individually in the stem 

or root extracts. 

Acetylcholinesterase inhibition (%) 

70 

60 

50 

40 

30 

20 

10 

0 

0.15625 mg/ml 

0.3125 mg/ml 

0.625 mg/ml 

cde 

bc 

de e 


Huernia hystrix. Bars bearing different letters are significantly 

different (P = 0.05) according to DMRT. The AChE inhibition 

activities by galanthamine at 0.5, 1.0 and 2 µM were 49.24, 59.81 

and 77.03%, respectively. 

de 

bcd 

Plant part 

bcde 

Stems Roots Whole plants 

b 

a 

114

5.3.2.5 Antioxidant activity 

5.3.2.5.1 DPPH radical scavenging activity 

Figures 5.5 and 5.6 show the dose-response radical scavenging activities 

observed in the MeOH extracts of different parts of Barleria species and Huernia 

hystrix, respectively. In all the extracts, there was an increase in the DPPH radical 

scavenging activity with increasing extract concentration. The dose-dependent 

curve of H. hystrix whole plant extract was however less steep when compared to 

its root or stem dose-dependent curves (Figure 5.6). From their dose-response 

activities, their EC50 values were obtained and presented in Tables 5.7 and 5.8. 

The EC50 values for the different extracts of Barleria speces ranged from 6.65 to 

12.56 µg/ml (Table 5.7). The EC50 values of B. greenii leaf and all B. prionitis 

extracts were in fact not significantly different from the EC50 of ascorbic acid, a 

standard antioxidant agent used as a positive control. In the case of B. greenii, the 

leaf extract showed a lower EC50 value compared to the stem and significantly, the 

root. This observation correlates well with AChE inhibitory activities observed in 

the leaves, stems and roots of B. greenii (Figure 5.3). According to HOUGHTON 

et al. (2007), free radical reactions are involved in the pathology of many diseases 

like Alzheimer‟s disease, cancer and inflammation. 

The stem and the root extracts of H. hystrix showed a good radical scavenging 

activity as indicated by their EC50 values, which were not significantly different 

from that of the ascorbic acid standard (Table 5.8). In the whole plant however, the 

radical scavenging activity was significantly lower than any of the individual plant 

parts. The results suggest the likely presence of certain compounds that are 

perhaps antagonistic in their radical scavenging activities. 

115

(A) 

DPPH Scavenging activity (%) 

(B) 


(C) 


110 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

110 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

B. greenii 

B. prionitis 

B. albostellata 

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 

0 

110 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

Log [Concentration (g/ml)] 


B. prionitis 


-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 

0 



B. prionitis 

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 



different parts of three Barleria species. (A) Leaves (B) Stems (C) 

Roots. 

116


100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 


H. hystrix roots 

H.hystrix stems 

H. hystrix whole plants 


different parts of Huernia hystrix. 

Table 5.7: DPPH radical scavenging activity of different parts of three Barleria 

species 

Plant species Plant part 

DPPH radical scavenging 

EC50 (µg/ml) R² 

B. prionitis Leaves 7.14 ± 0.056 bcd 0.9949 

Stems 6.65 ± 0.037 bc 0.9965 

Roots 6.94 ± 0.033 bc 0.9984 

B. greenii Leaves 7.24 ± 0.326 bcd 0.9881 

Stems 8.09 ± 0.266 d 0.9954 

Roots 12.56 ± 0.401 e 0.9307 

B. albostellata Leaves 7.52 ± 0.169 cd 0.9952 

Ascorbic acid 

Stems 7.31 ± 0.175 cd 0.9973 

6.17 ± 0.434 b 0.9550 

Butylated hydroxytoluene 3.75 ± 0.764 a 0.9027 

Mean values followed by different letters are significantly different (P = 0.05) 

according to DMRT. 

117

Table 5.8: DPPH radical scavenging activity of different parts of Huernia hystrix 

Plant part 

DPPH radical scavenging 

EC50 (µg/ml) R² 

Stems 7.01 ± 0.183 a 0.9805 

Roots 7.63 ± 0.154 a 0.9724 

Whole plants 393.83 ± 7.364 b 0.9542 

Ascorbic acid 6.17 ± 0.434 a 0.9550 

Butylated hydroxytoluene 3.75 ± 0.764 a 0.9027 

Mean values followed by different letters are significantly different (P = 0.05) 


5.3.2.5.2 Ferric ion reducing power activity 

The ferric ion reducing power assay (FRAP) is an assay based on an electron 

transfer reaction (HUANG et al., 2005). In the assay, the presence of reductants 

(antioxidants) in the tested extracts results in the reduction of the ferric 

ion/ferricyanide complex to the ferrous form, with a characteristic formation of 

Perl‟s Prussian blue, which is measured spectrophotometrically (CHUNG et al., 

2002). The degree of colour change is directly proportional to the antioxidant 

concentrations in the extracts (HUANG et al., 2005). 

Figures 5.7 and 5.8 show the reducing power of different extracts of three Barleria 

species and H. hystrix, respectively. All the extracts evaluated showed an increase 

in reducing power activity with an increase in extract concentration. However, the 

reducing activities of the extracts were significantly lower than the ascorbic acid 

and BHT standard controls. The ferric reducing power activity of the leaf and stem 

extracts of both B. prionitis and B. greenii were significantly higher than that of 

their root extracts (Figure 5.7), indicating a potential plant part substitution of the 

leaves or stems for the roots. 

118

(A) 

Absorbance 630nm 

(B) 


(C) 


2.5 

2.0 

1.5 

1.0 

0.5 

0.0 

0 10 20 30 40 50 60 70 80 

Concentration (g/ml) 

2.5 

2.0 

1.5 

1.0 

0.5 

0.0 

0 10 20 30 40 50 60 70 80 


2.5 

2.0 

1.5 

1.0 

0.5 

0.0 

0 10 20 30 40 50 60 70 80 



B. prionitis 


BHT 

Ascorbic acid 


B. prionitis 


BHT 

Ascorbic acid 


B. prionitis 

BHT 

Ascorbic acid 

Figure 5.7: Ferric ion reducing power activity of different parts of Barleria 

species. (A) Leaves (B) Stems (C) Roots. 

119

Absorbance 630 nm 

2.2 

2.0 

1.8 

1.6 

1.4 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

0 10 20 30 40 50 60 70 80 


H. hystrix roots 

H. hystrix stems 

H. hystrix whole plants 

BHT 

Ascorbic acid 

Figure 5.8: Ferric ion reducing power activity of different parts of Huernia 

hystrix. 

As shown in Figure 5.8, all the extracts of H. hystrix demonstrated a poor ferric 

reducing power activity compared to either of the standard controls (ascorbic acid 

and BHT) or even the Barleria species. There was no significant difference in the 

ferric reducing power activity demonstrated by the different parts evaluated. Taken 

together however, the results suggest the presence of antioxidant compounds with 

electron-donating ability in the different plant parts evaluated, which the assay is 

known to measure semi-quantitatively (AMAROWICZ et al., 2004; RUMBAOA et 

al., 2009). The presence of these compounds perhaps in an impure form or in 

small amounts in the extracts may be responsible for the generally low activity 

demonstrated by the extracts. 

5.3.2.5.3 β-Carotene linoleic acid assay 

This assay involves the heat-induced bleaching of carotenoids and is based on 

hydrogen atom transfer reactions (HUANG et al., 2005). According to 

AMAROWICZ et al. (2004), the abstraction of a hydrogen atom from linoleic acid 

120

during oxidation results in the formation of a pentadienyl free radical. This free 

radical subsequently attacks the highly unsaturated β-carotene molecules, leading 

to the loss of conjugation of the β-carotene molecules and the characteristic 

orange colour of the carotenoids (AMAROWICZ et al., 2004). The presence of 

antioxidants, capable of donating hydrogen atoms can however prevent or reduce 

carotenoid bleaching by quenching free radicals formed within the system 

(BURDA and OLESZEK, 2001; AMAROWICZ et al., 2004). 

The antioxidant activities of different parts of three Barleria species and H. hystrix 

in the β-carotene-linoleic acid model system are shown in Figures 5.9 and 5.10, 

respectively. In Barleria species, the antioxidant activity based on the average β- 

carotene bleaching rate ranged from 52 to 77%. The antioxidant activity of B. 

prionitis roots was significantly higher than that of its leaves and stems (Figure 

5.9A). There was no significant difference observed in the antioxidant activity 

(based on average β-carotene bleaching rate) of the different parts of either B. 

greenii or B. albostellata evaluated. This finding again suggests the potential to 

substitute the leaves of B. greenii for its stems or roots. 

The same trend recorded in the antioxidant activity of the extracts (based on the 

average β-carotene bleaching rate) was also observed with their oxidation rate 

ratio (Figure 5.9B). In this case, the lower the oxidation rate ratio, the higher the 

antioxidant activity of the different plant parts. According to AMAROWICZ et al. 

(2004), the antioxidant activity at 60 or 120 min possibly demonstrates the 

antioxidant activity of an extract more accurately than the oxidation rate ratio or 

antioxidant activity based on the average β-carotene bleaching rate. With the 

exception of B. prionitis stem, B. greenii stem and B. albostellata leaf extracts, the 

same trend observed for antioxidant activity at 60 min (Figure 5.9C) was also 

recorded at 120 min for all the extracts (Figure 5.9D). There was no significant 

difference between the antioxidant activities of B. greenii leaves and roots as well 

as the BHT standard control at either t = 60 or 120 min. BHT is one of the 

synthetic antioxidants mostly used as a food preservative (HASSAS-ROUDSARI 

et al., 2009). The toxicity of these synthetic antioxidants has however raised 

concern about their health safety, resulting in the increased search for naturally 

occurring antioxidants useful in food and cosmetic industries and as nutraceuticals 

121

(ORHAN et al., 2009; ABDEL-HAMEED, 2009). The findings from this current 

study suggest that the leaves and roots of B. greenii possibly contain antioxidant 

agents with activity equivalent to that of BHT, which can potentially be exploited as 

alternatives in the food and cosmetic industries. The purification of the antioxidant 

agents present in these plant materials could perhaps improve their antioxidant 

capacity. The use of the leaves of B. greenii is more sustainable and can 

potentially substitute for the roots, owing to their equivalent antioxidant activity in 

this assay. 

ANT (%) 

Antioxidant activity (%) at 60 min 

100 

80 

60 

40 

20 

0 

80 

60 

40 

20 

0 

A 

C 

e 

b 

bcd 

ab 

cde 

ab 

B. prionitis 



BHT 

bc 

cde 

de 

ab ab 

ab 

ab 

a 

bc 

ab 

a 

a 

0 

Leaves Stems Roots Control Leaves Stems Roots Control 

Plant part 

Plant part 

Figure 5.9: Antioxidant activities of different parts of three Barleria species in β- 

carotene-linoleic acid model system. Bars bearing different letters in each 

graph are significantly different (P = 0.05) according to DMRT. (A) 

Antioxidant activity (ANT) based on the average β-carotene bleaching 

rate. (B) Oxidation rate ratio (ORR). (C) Antioxidant activity (AA) at t = 60 

min. (D) Antioxidant activity (AA) at t = 120 min. 

Oxidation rate ratio 


0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0.0 

60 

40 

20 

B 

D 

d 

bc 

bc 

cd 

abc ab 

d 

abc 

bcd 

c 

bc 

a 

ab 

a 

bc 

abc 

a 

a 

122

In H. hystrix, the roots consistently showed a significantly higher antioxidant 

activity (based on average β-carotene bleaching rate), oxidation rate ratio as well 

as antioxidant activity at either 60 or 120 min, compared to the stems or whole 

plant (Figure 5.10A-D). Although there was no significant difference between the 

antioxidant activity of the roots and the standard synthetic antioxidant (BHT) at 60 

or 120 min, the use of the roots of this plant may not be sustainable from a 

conservation point of view. In general however, the activities recorded in the 

different parts of all the species evaluated in this study indicate the presence of 

antioxidant compounds with the ability to donate hydrogen atoms. 

ANT (%) 


100 

80 

60 

40 

20 

0 

80 

60 

40 

20 

0 

A 

Stems 

Roots 

Whole plants 

a 

0.8 

B 

BHT 

b 

0.6 

c 

c 

Figure 5.10: Antioxidant activities of different parts of Huernia hystrix in β-carotene-linoleic 

Oxidation rate ratio 

0.0 

C D 

a 

a 

b 

b 

Plant part 


acid model system. Bars bearing different letters in each graph are 

significantly different (P = 0.05) according to DMRT. (A) Antioxidant activity 

(ANT) based on the average β-carotene bleaching rate. (B) Oxidation rate 

ratio. (C) Antioxidant activity at 60 min. (D) Antioxidant activity at 120 min. 

0.4 

0.2 

60 

40 

20 

0 

c 

b 

b 

a 

c 

b 

Plant part 

a 

a 

123

5.3.3 Phytochemical evaluation 

5.3.3.1 Total phenolic content 

Figure 5.11 presents the total phenolic content of different parts of three Barleria 

species. In all three species, the highest total phenolic content was observed in 

the leaves, compared to other plant parts. In B. prionitis and B. greenii, the stem 

phenolic contents were significantly higher than that of the root. The highest (12.79 

mg GAE/g DW) and lowest (1.95 mg GAE/g DW) total phenolic content were 

recorded in B. prionitis leaves and B. greenii roots, respectively. 

Figure 5.12 shows the total phenolic content of different parts of H. hystrix. The 

roots had the highest total phenolic content (181.22 µg GAE/g), although not 

significantly different from that of the stems. The total phenolic content of the 

whole plant was the lowest with 128.73 µg GAE/g DW. 

Phenolic compounds include flavonoids, condensed tannins and hydrolysable 

tannins, many of which are reported to have antimicrobial, anti-inflammatory and 

antioxidant activities (MARCUCCI et al., 2001; POLYA, 2003). According to 

SAMAK et al. (2009), the redox property of phenolic compounds is an important 

underlying factor for their antioxidant activity, giving them the ability to act as 

hydrogen donors, reducing agents and singlet oxygen quenchers. In the light of 

the total phenolic content recorded in the different parts of the plant species in the 

current study, the amounts of some particular groups of phenolic compounds were 

further evaluated in the different parts of the studied species. This could possibly 

help in underpinning the specific phenolic groups likely to be responsible for the 

observed pharmacological activities. 

124

Total phenolic content 

(mg GAE/g DW) 

Figure 5.11: Total phenolic content of different parts of three Barleria species. 

Total phenolic content 

(µg GAE/g DW) 

14 

12 

10 

8 

6 

4 

2 

0 

250 

200 

150 

100 

50 

0 

a 

b 

e 


Plant part 

Bars bearing different letters are significantly different (P = 0.05) 


ab 

Figure 5.12: Total phenolic content of different parts of Huernia hystrix. Bars 

bearing different letters are significantly different (P = 0.05) 


c 

f 

a 

f 

Plant part 

b 

d 

B. prionitis 



g 

Stems 

Roots 

Whole plants 

125

5.3.3.2 Total iridoid content 

Figure 5.13 shows the total iridoid content of different parts of the three Barleria 

species. The highest total iridoid content was recorded in B. albostellata leaves 

with 801.4 µg HE/g DW while the lowest was found in B. prionitis roots with 30 µg 

HE/g DW. In each of the Barleria species, the total iridoid content of the leaves 

was significantly higher than that of the other parts. There was no significant 

difference between the iridoid contents of the stems of the three species. The total 

iridoid content of B. greenii roots was significantly higher than that of B. prionitis 

roots. The isolation of six known iridoids (shanzhiside methyl ester, 6-O-trans-p- 

coumaroyl-8-O-acetylshanzhiside methyl ester, barlerin, acetylbarlerin, 7- 

methoxydiderroside and lupulinoside) with different levels of AChE inhibitory and 

free radical scavenging activities from aerial parts of B. prionitis has been reported 

(ATA et al., 2009). The AChE inhibitory and radical scavenging activities recorded 

in the Barleria species evaluated in this study could be due to the presence of 

these or other iridoid compounds. The presence of iridoid compounds even at a 

low concentration in all parts of the three Barleria species could perhaps play a 

role in the pharmacological activities observed in all the extracts evaluated. Since 

the leaves of each of these Barleria species contained more iridoid compounds, 

two- to twelve-fold higher than their stems or roots, they may possibly be a 

potential sustainable source for iridoid compounds. 

In H. hystrix, the roots had the highest total iridoid content of 92.6 µg HE/g DW 

(Figure 5.14). This value was however, not significantly different from the total 

iridoid content of the stems or whole plant. Some iridoids or iridoid-rich plant 

extracts have been reported to demonstrate antimicrobial and anti-inflammatory 

activities (BOLZANI et al., 1997; WANIKIAT et al., 2008). The pharmacological 

activities observed in the studied species could perhaps be attributed in part, to 

the iridoids present in the different parts of this species. 

126

Total iridoid content 

(µg HE/g DW) 

Figure 5.13: Total iridoid content of different parts of three Barleria species. Bars 

Total iridoid content 

(µg HE/g DW) 

1000 

800 

600 

400 

200 

120 

100 

80 

60 

40 

20 

0 

0 

d 

b 

a 


Plant part 



Figure 5.14: Total iridoid content of different parts of Huernia hystrix. Bars 



a 

e e e 

a 

Plant part 

a 

f 

c 

B. prionitis 



Stems 

Roots 

Whole plants 

127

5.3.3.3 Flavonoid content 

The flavonoid content of different parts of the three Barleria species is presented in 

Figure 5.15. The highest (3.92 mg Catechin/g DW) and lowest (0.35 mg 

Catechin/g DW) flavonoid contents were recorded in B. greenii leaves and B. 

albostellata stems, respectively. In general, higher flavonoid content was observed 

in the leaves compared to the other plant parts in each of the species. 

Figure 5.16 shows the flavonoid content of different parts of H. hystrix. Although 

there was no significant difference between the values, the flavonoid content of the 

roots was higher than that of the whole plant or stems. 

In an extensive review on the effects of naturally occurring flavonoids on 

mammalian cells, MIDDLETON et al. (2000) observed that flavonoids 

demonstrate a noteworthy array of biochemical and pharmacological actions, most 

notable being their antioxidant, anti-inflammatory and antiproliferative effects. 

Many other researchers have reported the antioxidant, anti-inflammatory and 

antimicrobial activities of flavonoids or flavonoid-rich extracts (BURDA and 

OLESZEK, 2001; HAVSTEEN, 2002; TUNALIER et al., 2007; PATTANAYAK 

and SUNITA, 2008). In the present study, the flavonoid content in all the different 

parts of the studied species appeared to be substantially higher compared to other 

phenolic groups evaluated in this study. It is therefore likely that the observed 

pharmacological activities in the different plant parts are largely due to their 

flavonoid content. In addition to their quantity however, the quality or nature of the 

flavonoid present in the different plant parts could make a difference in their 

therapeutic potential. 

128

Flavonoid content 

(mg Catechin/g DW) 

Figure 5.15: Flavonoid content of different parts of three Barleria species. Bars 

Flavonoid content 

(mg Catechin/g DW) 

5 

4 

3 

2 

1 

0 

2.0 

1.8 

1.6 

1.4 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

b 

a 




Figure 5.16: Flavonoid content of different parts of Huernia hystrix. Bars bearing 


DMRT. 

c 

a 

c 

c 

c 

Plant part 

a 

Plant part 

a 

bc 

B. prionitis 



b 

Stems 

Roots 

Whole plants 

129

5.3.3.4 Gallotannin content 

The roots of B. greenii showed the highest gallotannin content with 145.33 µg 

GAE/g DW compared to all other Barleria species parts (Figure 5.17). In all three 

species, the gallotannin content of the leaves was significantly higher than that of 

the stems. The gallotannin contents of the leaves and stems of B. albostellata 

were significantly higher than that of the leaves and stems, respectively of other 

Barleria species. 

Figure 5.18 shows the gallotannin content of the different parts of H. hystrix. The 

stems had a higher gallotannin content (49.05 µg GAE/g DW) compared to the 

roots and the whole plant. There was however, no significant difference between 

the values obtained. 

TIAN et al. (2009a) reported antioxidant and antibacterial activities of gallotannin- 

rich extracts from Galla chinensis. ENGELS et al. (2009) also reported 

antimicrobial activities of gallotannin-rich extracts and gallotannins isolated from 

Mangifera indica kernels. The antibacterial activity of hydrolysable tannins (the 

group to which gallotannins belong) isolated from medicinal plants used in treating 

gastric disorders against Helicobacter pylori has been demonstrated 

(FUNATOGAWA et al., 2004). Some gallotannins have also been reported to act 

as inhibitors of particular enzymes such as the COX enzymes involved in the 

inflammatory pathway (POLYA, 2003). The presence of gallotannins in different 

parts of the species evaluated in this study could, at least, partly contribute to their 

antioxidant, anti-inflammatory and antibacterial activities. 

130

Gallotannin content 


Figure 5.17: Gallotannin content of different parts of three Barleria species. Bars 

Gallotannin content 


160 

140 

120 

100 

60 

50 

40 

30 

20 

10 

80 

60 

40 

20 

0 

0 

a 

b 

b b 

Plant part 



Figure 5.18: Gallotannin content of different parts of Huernia hystrix. Bars 



d 

B. prionitis 



d 


a 

a 

Plant part 

a 

c 

a 

Stems 

Roots 

Whole plants 

131

5.3.3.5 Condensed tannin (proanthocyanidin) content 

Proanthocyanidins are made up of oligomeric and polymeric flavan-3-ols, and they 

have been reported to demonstrate strong antioxidant capacity (SHYU et al., 

2009). SHAN et al. (2007) reported the significant contribution of proanthocyanidin 

to the antibacterial activity of Cinnamomum burmannii. The condensed tannin 

content of different parts of the three Barleria species evaluated in this study is 

presented in Figure 5.19. No condensed tannin was detected in B. prionitis leaves 

and B. albostellata stems. Where condensed tannin was recorded, the content 

was generally low (ranging from 0.007 to 1.2% per g DW) in all parts of the three 

Barleria species evaluated. All the different parts of B. greenii had a significantly 

higher proanthocyanidin content compared to any part of B. prionitis and B. 

albostellata. There was no condensed tannin detected in H. hystrix. These findings 

indicate that the pharmacological activities recorded in different parts of H. hystrix 

as well as B. prionitis leaves and B. albostellata stems could not be due to 

proanthocyanidins. In other parts of Barleria species however, their 

proanthocyanidin content could possibly contribute, at least to a small extent, to 

their antioxidant and antibacterial activities. 

Condensed tannin 

(% per g DW) 

1.4 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

a 

e 

e e e 


c 

Plant part 

B. prionitis 



Figure 5.19: Condensed tannin content (as leucocyanidin equivalents) of 

d 

b 

different parts of three Barleria species. Bars bearing different 

letters are significantly different (P = 0.05) according to DMRT. 

132

Overall, the results from this study demonstrate the therapeutic potential of the 

Barleria species evaluated as well as that of H. hystrix. As far as can be 

ascertained and besides the published article from this study, this is the first report 

on the antimicrobial, anti-inflammatory, AChE inhibition and antioxidant activities of 

B. greenii, B. albostellata and H. hystrix. The observed activities might largely be 

due to their flavonoid content, with a contributing effect from their iridoid and tannin 

compounds. The concept of substituting plant parts for sustainable exploitation 

appeared to be dependent on the species and/or biological activity evaluated. The 

substantial better activities observed with B. greenii in some cases highlight the 

need to conserve our plant resources before they become extinct, since some of 

them could be pharmacologically active and perhaps contain novel compounds 

that are biologically active against some treatment-resistant infections. 

133

Chapter 6 General conclusions 

An effective micropropagation protocol was developed for B. greenii, an 

endangered and endemic South Africa horticultural shrub. Based on the highest 

regeneration rate obtained, more than 60,000 transplantable shoots per year can 

potentially be produced from a single shoot-tip explant using this protocol. 

Culturing under a 16 h photoperiod was more favourable for adventitious shoot 

production than culturing under continuous light. Higher adventitious shoot 

production was obtained in the treatments with BA alone when compared to the 

BA treatments supplemented with NAA concentrations. These results indicate that 

the exogenous application of NAA is not a requirement for adventitious shoot 

production of this plant species. The treatments with BA, commonly used in 

micropropagation industry, resulted in a comparatively high abnormality index in 

regenerated adventitious shoots from shoot-tip explants. Increased adventitious 

shoots and reduced abnormality indices were observed in the treatments with 

mTR and MemTR even at higher concentrations (5 and 7 µM). The topolins (mTR 

and MemTR) are less toxic and more effective than BA in the micropropagation of 

this plant species. The abnormality indices recorded in treatments with some 

topolin concentrations were in fact lower than what was observed in the control. It 

is possible that the observed abnormalities in the treatments with topolins are 

carry-over effects of BA since the explants used were taken from BA-treated 

cultures. The use of explants that have not been exposed to BA treatments may 

be necessary to confirm the effect of the topolin treatments on the induction of 

abnormalities in this plant species. 

Unlike in B. greenii, the treatments with a combination of BA and NAA showed a 

synergistic effect on adventitious shoot regeneration of H. hystrix, an endangered 

ornamental succulent. Regenerated adventitious shoots were successfully 

acclimatized with a high survival rate. Environmental factors such as temperature 

and photoperiod significantly affected adventitious shoot production. This study 

highlights the need to investigate the effects of environmental conditions when 

developing efficient micropropagation protocols, especially for commercial 

purposes. Optimizing environmental conditions could increase shoot production, 

134

educe labour costs and thus subsequent production costs. The observations from 

this study also provide an insight into the physiology of H. hystrix when cultured at 

different temperatures and photoperiods. The results suggest that this ornamental 

succulent possibly has the ability to shift between the C-3 and CAM photosynthetic 

pathways depending on the photoperiod conditions under which the plants are 

cultured. In cultures maintained at lower temperatures (15 and 20°C) under a 16 h 

photoperiod, low shoot proliferation rates were observed due to slow growth and 

less differentiation of shoot meristems. In addition to offering an approach for 

short-term in vitro storage of H. hystrix germplasm, such slow growth at low 

temperatures is economically beneficial when manpower requirement for 

subculturing is not available. 

The pharmacological activities and phytochemical properties of the species 

studied in this research highlight their therapeutic potential. Extracts from different 

parts of three Barleria species and H. hystrix demonstrated different levels of 

antibacterial, antifungal, antioxidant, anti-inflammatory and AChE inhibition 

activities. The pharmacological activities observed in some extracts of H. hystrix 

might possibly explain its heavy exploitation in traditional medicine. In general, 

however, the Barleria species evaluated showed better pharmacological activities 

compared to H. hystrix. Although B. greenii has no recorded usage in traditional 

medicine, some of its extracts particularly demonstrated higher pharmacological 

activities in some cases than other Barleria species evaluated. In some of the 

pharmacological assays, the leaves of Barleria species and stems of H. hystrix 

demonstrated higher activities than the other plant parts, suggesting their potential 

in plant part substitution. The harvesting of leaves or stems as a conservation 

strategy is certainly more sustainable than the destructive use of the roots of these 

threatened plant species. The results obtained from this study also suggest that 

the concept of plant part substitution is dependent on the plant species and/or 

pharmacological activity of interest. The phytochemical evaluation of the studied 

species indicated that the various activities shown by the extracts could possibly 

be due to their phenolic (including flavonoids, proanthocyanidins and gallotannin) 

and iridoid content. The isolation of specific bioactive compounds through 

bioassay-guided fractionation and their characterization as well as studies 

135

evaluating their safety may be necessary in the exploration of these species for 

potential new therapeutic drugs or drug leads. 

Taken together, the present study highlights the need for the conservation of our 

indigenous plant resources. In vitro propagation methods offer powerful 

techniques for rapid propagation and germplasm storage of our endemic and 

threatened plant species. 

136

References 

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